Photoelectrocatalytic-microbial biohybrid for succinic acid synthesis

Domain: chemistry
Published: 2026-02-24
Editorial outcome: minor_revision

Abstract
Succinic acid is an important platform chemical traditionally produced via energy-intensive and environmentally unfriendly processes. Actinobacillus succinogenes offers a sustainable biosynthetic route, yet its productivity is constrained by limited intracellular electron transfer. Here, we develop a photoelectrocatalytic–microbial biohybrid system to overcome these metabolic bottlenecks. Adaptive laboratory evolution using gold nanoparticles establishes an enhanced charge-transfer pathway in Actinobacillus succinogenes, which is subsequently immobilized on a layer-by-layer NiO@PAA@NHS (NiO nanosheets coated with hydrogel of polyacrylic acid (PAA) grafted with N-Hydroxysuccinimide (NHS)) photoelectrode to construct a NiO@PAA@NHS/Au@Actinobacillus succinogenes biohybrid. Under simulated solar illumination at −0.3 V vs. RHE, the system delivers a photocurrent density of 1.9 mA cm-2, a CO2 conversion efficiency of 67%, and a succinic acid production rate of 1.41 ± 0.04 g L-1 h-1 cm-2. This work demonstrates an effective strategy for coupling solar energy with microbial metabolism for scalable, carbon-neutral chemical production.

Introduction
Succinic acid, alternatively referred to as amber acid or 1,4-butanedioic acid, is a promising raw material to produce 1,4-butanediol, γ-butyrolactone, tetrahydrofuran, 2-pyrrolidinone, and polybutylene succinate1,2. This versatile compound is widely applied across industries, including chemical synthesis, food flavoring and preservation, pharmaceutical manufacturing, and the development of biodegradable plastics3,4. Therefore, it is listed as one of the twelve high-value chemicals by the U.S. Department of Energy5. The market for succinic acid is anticipated to experience a compound annual growth rate of 10.6%, and it is projected that by the year 2026, the market will surpass the value of 200 million US dollars6. Currently, most of commercially available succinic acid is produced by chemical process, in which liquefied petroleum gas or maleic anhydride is used as a starting material7,8. However, the process poses several environmental and health concerns due to the release of greenhouse gases and pollutants, and the by-products generated in this process are toxic and require careful disposal9. Therefore, there is a growing interest in developing more sustainable and environmentally friendly alternatives to the current chemical production methods of succinic acid.

Recent analysis has shown that bacterial fermentative production of succinic acid from renewable resources can be more cost-effective than the petroleum-based processes10–12. Many different microorganisms have been screened and studied for succinic acid production13–15. Among them, Actinobacillus succinogenes (A. succinogenes), a naturally succinic acid-producing strain isolated from bovine rumen, is one of the most intensively investigated bacterium for succinic acid production16. The A. succinogenes has a natural capacity to convert both pentose and hexose sugars into succinic acid as an intermediate of the tricarboxylic acid (TCA) cycle17. Upon the absorption of the substrate glucose by the bacteria, it is converted into phosphoenolpyruvate (PEP), marking the beginning of the metabolic divergence into two distinct pathways18,19. One pathway is dedicated to the production of the desired product, succinic acid, and is primarily governed by the enzyme phosphoenolpyruvate carboxykinase (Pepck). Under the catalysis of PEPCK and with the aid of coenzymes such as NADH, CO2 is incorporated into the metabolic pathway. PEP undergoes a series of enzymatic transformations, initially forming oxaloacetate, which is then reduced to malate through the action of malate dehydrogenase, utilizing NADH. Subsequently, malate is converted to fumarate by fumarase, and finally, fumarate is transformed into succinic acid, the target product, in a reaction catalyzed by fumarate reductase. The other metabolic route, dominated by pyruvate kinase (PK), is responsible for the synthesis of by-products such as formic acid, acetic acid, and ethanol. This metabolic flexibility allows the bacteria to adapt to varying environmental conditions and substrate availabilities, optimizing the overall efficiency of succinic acid production within the PEC-bacterial biohybrid system. This metabolic pathway ingeniously incorporates CO2 20–22, rendering it an optimal candidate for transforming lignocellulosic sugars and CO2 into bio-products from sustainable feedstocks, and providing an additional advantage of contributing to the carbon peaking and carbon neutrality goals23–26. However, the inherent metabolic processes of microorganisms in the context of fermentation-based chemical synthesis impose constraints on the potential for increased product yields. Presently, two strategies have been posited to surmount these limitations and to augment production metrics. The first strategy encompasses genetic engineering, leveraging targeted modifications to refine and optimize metabolic routes27,28. However, compared to commonly industrial microorganisms, the repertoire of genetic modification techniques for A. succinogenes is rather confined, which hinders the rate of strain improvement through genetic engineering. The alternative paradigm introduces the assembly of biohybrids, which entails the amalgamation of nanomaterials with bacterial systems to supply the microorganisms with surplus electrons and energy, consequently elevating production levels29–31. Consequently, in response to the growing demand for sustainable and environmentally friendly production methods, the engineering of biohybrids emerges as an innovative and exceptionally prospective pathway for enhancing succinic acid yields. While this approach needs to address the critical requirements for sustainable energy input, efficient charge transfer, and intracellular electron transport, which are essential for enhancing succinic acid yields.

The photoelectrocatalytic-bacterial (PEC-bacterial) biohybrid system is a good candidate to harness sustainable solar energy and to drive the production of succinic acid from CO2 and renewable resources. This system integrates a photoelectrode with A. succinogenes, where the photoelectrode generates electrons upon illumination, and these electrons are transferred to the bacteria, facilitating the assimilation of CO2 into succinic acid. As the most active bacteria at the biotic-abiotic interface are those directly connected to the photoelectrode, and close microbial-electrode interactions can promote electron transport and CO2 reduction efficiency32,33. Therefore, the effective integration of the photoelectrode with the bacteria is the prerequisites for efficient charge transfer in the PEC-bacterial system. We draw inspiration from several innovative studies, for example, Zhang34 has designed 3D printed hierarchical column array electrodes that significantly boost bacterial loading efficiency. Additionally, Erwin Reisner35 has proposed an IO-ITO electrode with a hydrophilic surface and porous structure, which facilitates bacterial affinity. Liu36 has also contributed to the field with an in-situ chemical reaction-mediated covalent localization strategy of bacteria, where thiolated bacteria bind to poly(disulfide)s-abundant mucin located at various tissue interfaces.

Herein, the photoelectrode is designed with a typical p-type semiconductor of NiO nanosheets, which is coated with hydrogel of poly acrylic acid (PAA) grafted with N-Hydroxysuccinimide (NHS) (NiO@PAA@NHS). Hydrogel is a highly water-containing, three-dimensional network-structured material with excellent biocompatibility and ability to load a range of biologically active substances, such as bacteria, which not only have good adhesion to the bacteria, but also promote bacterial colonization and activity over a long period of time37,38. Furthermore, to ensure efficient extracellular and intracellular electron transport, the gold nanoparticles (Au NPs) are incubated into bacteria through an adaptive laboratory evolution (ALE) strategy, pioneered by Prof. Peidong Yang39, to form a conductive bridge that allows electrons generated extracellularly to be transported across the bacterial membrane and into the metabolic pathways of the bacteria. Moreover, the Au-ALE process can optimize the bacteria’s electroactivity, enhancing their ability to acquire and transfer electrons, which in turn improves interfacial charge transport while maintaining bacterial vitality40. Therefore, the PEC-bacterial biohybrid of NiO@PAA@NHS/Au@A. succinogenes provides necessary metabolic energy to boost succinic acid production, and reach a high succinic acid yield rate of 1.41 ± 0.04 g L−1 h−1 cm−2 (0.012 ± 0.0003 mol mol L−1 h−1 cm−2) at one atmospheric pressure saturated with CO2 under illumination of solar light. This PEC-bacterial biohybrid is expected to be applied in large-scale, low-cost succinic acid production, providing another direction for CO2 reduction, and sustainably producing high-value long-chain hydrocarbons. This innovative approach leverages solar energy and biohybrid technology to produce high-value chemicals in a sustainable and environmentally friendly manner.

Results
Construction of the PEC-Bacterial Biohybrid System
The PEC-bacterial biohybrid reactor, as schematically illustrated in Fig. 1a, is designed with a NiO@PAA@NHS/Au@A. succinogenes biohybrid photoelectrode to leverage the metabolic capabilities of A. succinogenes for the synthesis of succinic acid from CO2 under solar light illumination. Figure 1b illustrates the interaction between the PAA@NHS hydrogel and the A. succinogenes cell membrane. The ester groups (C=O) on the hydrogel surface interact with the amino groups (N-H) of membrane proteins through possible amide linkage formation, promoting robust bacterial adhesion on the photoelectrode surface. However, the current spectroscopic evidence only indicates interfacial coupling rather than definitive covalent bond formation. Notably, these bacteria have been adapted after Au-adaptive laboratory evolution (ALE) process, introducing Au NPs near the inner cell membrane. This strategic introduction of Au NPs establishes an efficient charge transfer pathway, enhancing the charge transfer efficiency and, consequently, increasing the yield of PEC production of succinic acid. This innovative approach highlights the potential of combining biocompatible materials with nanoscale engineering to optimize bio-photoelectrochemical systems.

Characterization of bacterial modification and surface immobilization
The Au NPs were introduced into A. succinogenes with an ALE strategy. As illustrated in Fig. 2a, this strategy enabled the bacteria to adapt to and tolerate the presence of these Au NPs. This process is essential for creating a biohybrid system where the bacteria can utilize the Au NPs for enhanced electron transfer. In the ALE process, A. succinogenes were initially exposed to a low concentration of Au ions, allowing the bacteria to encounter the Au ions without causing immediate toxicity. The Au ions were taken up by the bacteria, possibly through active transport mechanisms or simple diffusion41. Once inside the cells, the Au ions are reduced to Au NPs by reductive substances present in the bacterial cells, such as NADH or other biomolecules with reducing properties42–44. As the bacteria are exposed to increasing concentrations of Au ions over time, they develop mechanisms to tolerate and manage the presence of these Au NPs. The Au-ALE allows the bacteria not only to tolerate but also to thrive in the presence of Au NPs, thus significantly enhancing their survival rates.

Figure 2b displays a thin-sectioned transition electron microscopy (TEM) image of wild A. succinogenes, where it can be observed that the morphology is intact and spherical. As shown in Fig. 2c, the ALE of A. succinogenes with Au ions has resulted in the bacteria embedding Au NPs within their cellular structure, particularly near the inner wall of the bacterial cell membrane. The size distribution of Au NPs in bacteria is shown in Supplementary Fig. 1, with an average particle size of approximately 38 nm. The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image in Fig. 2d reveals distinct bright spots, which, according to this characterization method, indicate a higher atomic number of an element. The brightness of the spots corresponds to the presence of elements with higher atomic numbers, and in this case, it further demonstrates the presence of Au NPs within the bacterial cells, located near the inner wall of the cell membrane. The corresponding energy-dispersive X-ray spectroscopy (EDS) mapping image in Fig. 2e for Au@A. succinogenes also confirms the successful entry of Au NPs into the bacterial cells. The EDS data show a higher concentration of Au near the cell membrane, suggesting that the Au NPs were not only internalized by the bacteria but also accumulated near the cell membrane, as evidenced by the intense EDS signal for Au in that region. This membrane-associated localization of Au NPs can be attributed to their intracellular formation mechanism. During the ALE process, A. succinogenes gradually adapted to increasing concentrations of HAuCl4, reducing Au3+ to Au0 likely using intracellular reductants such as NADH and NADH-dependent reductases. Since many of these reductases are enriched at or near the inner membrane, the nucleation of Au0 preferentially occurs in that region, resulting in the accumulation of Au NPs adjacent to the membrane. Although Fig. 2e shows the overall Au distribution, this proximity to the membrane is further supported by HAADF-STEM and EDS mapping. Such spatial positioning is essential for the Au NPs to serve as intracellular conduits for electron transfer, bridging the photoelectrode-derived electrons with bacterial metabolic pathways without disrupting cellular viability.

Despite the presence of these Au NPs, we have observed that the bacteria continue to divide (Supplementary Fig. 2). Moreover, the uniform distribution of Au NPs within each bacterium further substantiates the successful complexation of the Au NPs and their excellent biocompatibility. Furthermore, we compared the viability of A. succinogenes cultured directly in a high-concentration Au ion solution (2 μM) versus those cultured using a stepwise acclimation approach (0.5 μM, 1 μM, 1.5 μM, and 2 μM) with flow cytometry (Supplementary Fig. 3). As shown in Fig. 2f, the apoptotic analysis revealed that 73.1% of the 100,000 bacteria treated directly with 2 μM HAuCl4 underwent apoptosis. In contrast, when bacteria were adapted to 2 μM HAuCl4, the survival rates surged to 89.1%. This significant difference demonstrates that the ALE approach is an effective way to introduce high concentrations of Au NPs within bacterial cells, which is crucial for enhancing charge transfer efficiency. All these micromorphological and elemental evidence provide compelling evidence of the intracellular localization of Au NPs, highlighting their potential role in facilitating electron transfer processes within the bacteria and potentially enhancing the overall efficiency of the PEC-bacterial biohybrid system.

The Au-ALE A. succinogenes bacteria are adsorbed onto the surface of photoelectrodes, which are prepared by coating a PAA@NHS hydrogel on NiO nanosheets (Fig. 2g). We performed FTIR spectroscopy to analyze the interfacial chemical environment (Supplementary Fig. 4). The free A. succinogenes spectrum shows distinct amide I (1627 cm−1) and amide II (1533 cm−1) bands45. After immobilization on NiO@PAA@NHS, new features emerge at 1685 cm−1 and 1625 cm−1 with significant attenuation of the amide II signal. This behavior suggests enhanced hydrogen bonding and possible amide-linkage interactions between bacterial surface proteins and the NHS-activated hydrogel layer46. The weakened amide II band is partly due to spectral overlap with the C=O stretching of the hydrogel (≈1710 cm−1) and the broad O-H/N-H bending envelope (1550–1650 cm−1), indicating that these shifts reflect interfacial coupling rather than definitive covalent bond formation. Additionally, the band at 1043 cm−1 is attributed to C–O–C stretching vibrations of polysaccharides present on the bacterial cell wall or C–O stretching in polyacrylic acid (PAA)47. After incubating the bacteria-with NiO@PAA@NHS, NiO@PAA, and bare NiO electrodes in 0.1 M PBS solution for 30 min, CLSM analysis (Supplementary Fig. 5) revealed negligible bacterial detachment from the NiO@PAA@NHS electrode, whereas NiO@PAA and bare NiO controls showed stronger fluorescence signals corresponding to higher bacterial release. It is likely that the NHS-activated hydrogel layer could improve interfacial adhesion and biofilm stability.

The NiO nanosheets, synthesized via a hydrothermal method on nickel foam, exhibit a distinct lamellar structure with a fine, fluffy nanostructure as observed in the SEM image (Supplementary Fig. 6). High-resolution TEM (HR-TEM) analysis reveals an interplanar spacing of 0.23 nm (Supplementary Fig. 7), corresponding to the (200) plane of NiO, confirmed by the XRD pattern (Supplementary Fig. 8).

The formation of the NiO@PAA@NHS sample was characterized using FTIR, Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). The FTIR analysis (Supplementary Fig. 9) shows peaks at 1710 cm−1, 1423 cm−1, and 1270 cm−1, corresponding to the stretching vibration of the C=O bond, the scissor bending vibration of -CH2, and the stretching vibration of C-N, respectively48. A new Raman peak (Supplementary Fig. 10) after encapsulating PAA@NHS can be assigned to C-H asymmetric stretching of PAA@NHS. The XPS data indicate a new N1s peak and variation of O1s peaks, evidencing the formation of the NiO@PAA@NHS material (Supplementary Fig. 11). The nanosheet structures are well-preserved after the PAA@NHS hydrogel coating, maintaining a predominantly rounded and smooth morphology (Supplementary Fig. 12). A TEM image further confirms the wrapping of PAA/NHS around the surface of the NiO nanosheets (Supplementary Fig. 13), and the crystal structure of NiO remains unchanged after the PAA@NHS coating, as shown by the XRD pattern (Supplementary Fig. 8). The elemental distribution on NiO@PAA@NHS is revealed by a HAADF-STEM image and corresponding EDS elemental mapping images (Supplementary Fig. 14), showing a uniform distribution of Ni, N, and O elements, with the nitrogen originating from the NHS component within the PAA@NHS hydrogel. Collectively, these results confirm the successful construction of the NiO@PAA@NHS photoelectrode.

After the successful fabrication of the NiO@PAA@NHS electrode, the A. succinogenes after the Au-ALE were adsorbed onto its surface. SEM analysis, as depicted in Fig. 2h, i, conducts a comparative assessment of bacterial cell counts and clearly illustrates a pronounced increase in bacterial colonization on the NiO@PAA@NHS photoelectrode surface. This increase indicates a marked enhancement in bacterial adhesion attributable to the presence of the PAA@NHS hydrogel layer. A quantitative summary of A. succinogenes bacteria is presented in Fig. 2j, showing an average bacterial count of 0.16 × 106/mm2 on pristine NiO. In contrast, this count significantly rose to 2.6 × 106/mm2 on the NiO@PAA@NHS surface. Such a substantial enhancement in bacterial adhesion highlights the critical role of surface functionalization in augmenting bacterial adhesion and metabolic activity within the PEC-bacterial biohybrid system. This surface modification is pivotal for creating a conducive environment for bacterial attachment and activity, thereby enhancing the overall performance of the biohybrid system.

Photoelectrocatalytic performance of the biohybrid system
The PEC activity of NiO@PAA@NHS/Au@A. succinogenes biohybrid was evaluated by linear sweep voltammetry (LSV) in an Ar- or CO2-saturated electrolyte, in the dark or under illumination of simulated solar light (AM1.5G, 100 mW cm−2). As depicted in Fig. 3a, in the Ar-saturated electrolyte, the NiO@PAA@NHS/Au@A. succinogenes biohybrid exhibited a weak photocurrent response. However, in the CO2-saturated electrolyte, the current densities both in dark and under illumination were significantly enhanced, indicating effective CO2 reduction involvement in succinic acid production. The succinic acid yields were compared under CO2 and Ar atmospheres with or without the addition of Mg(OH)2CO3. As a pH neutralizer, the Mg(OH)2CO3 also served as an additional carbon source during the reaction process. Therefore, when Ar was introduced without the addition of basic magnesium carbonate, A. succinogenes essentially produced no succinic acid (Supplementary Fig. 15). Furthermore, the current density on the NiO@PAA@NHS/Au@A. succinogenes biohybrid under illumination was much higher than that in the dark, suggesting more effective CO2 reduction and a higher succinic acid yield in the PEC model than that in the electrocatalytic (EC) model. These observations underscore the system’s capacity for CO2 reduction and its efficient harnessing of photogenerated electrons, thereby enhancing succinic acid production.

The succinic acid yield rate of the NiO@PAA@NHS/Au@A. succinogenes biohybrid was evaluated under various operational models, revealing significant differences in performance. As presented in Fig. 3b, the PEC model (PEC-biohybrid) demonstrated the highest yield rate of 1.41 ± 0.04 g L−1 h−1 cm−2 (0.012 ± 0.0003 mol mol L−1 h−1 cm−2), showcasing its superior efficiency in succinic acid production. This outcome underscores the effectiveness of integrating light-based energy input to enhance bacterial metabolism and succinic acid generation. The EC model (EC-biohybrid) followed with a yield rate of 0.75 ± 0.03 g L−1 h−1 cm−2, while the photocatalytic model (PC-biohybrid) exhibited a rate around 0.58 ± 0.06 g L−1 h−1 cm−2. In contrast, the biohybrid under dark and open circuit conditions had the lowest yield rate of 0.21 ± 0.04 g L−1 h−1 cm−2. These results suggest that external energy input, particularly through the PEC model, can substantially increase the rate of succinic acid production by A. succinogenes, highlighting the potential of this approach in bioprocess optimization and sustainable chemical production.

Figure 3c provides a comparative analysis of the succinic acid yield rate across four biohybrid systems, including NiO@PAA@NHS/Au@A. succinogenes, NiO@PAA/Au@A. succinogenes, NiO/Au@A. succinogenes, and Au@A. succinogenes. The NiO@PAA@NHS/Au@A. succinogenes biohybrid system reaches the highest yield rate. The NiO@PAA/Au@A. succinogenes system also performs well, with a yield rate of 0.98 ± 0.06 g L−1 h−1 cm−1. In contrast, the NiO/Au@A. succinogenes, and the Au@A. succinogenes, which lack the additional components, show lower yield rates of just 0.47 ± 0.03 g L−1 h−1 cm−1 and 0.21 ± 0.02 g L−1 h−1 cm−1, respectively. Furthermore, the CO2 conversion efficiency (Supplementary Fig. 16) and glucose conversion rate (Supplementary Fig. 17) were systematically analyzed across different systems. The NiO@PAA@NHS/Au@A. succinogenes biohybrid system demonstrated superior performance, achieving a remarkable 67% CO2 conversion rate along with a glucose conversion yield of 0.78 g/g glucose. These results underscore the importance of the biohybrid composition in maximizing succinic acid yield, with the NiO@PAA@NHS/Au@A. succinogenes system demonstrating the most promising results due to the contribution of Au NPs acting as an efficient charge transfer bridge and the hydrogel’s effective adhesion to bacteria.

The photocurrent densities at different applied potentials of NiO@PAA@NHS/Au@A. succinogenes biohybrid was compared and is presented in Fig. 3d. The biohybrid shows stable photocurrent performance, and the photocurrent density increases with more negative potentials, indicating efficient charge separation and transport at the PEC-bacterial model. In order to further screen the appropriate potential for succinic acid production, we comprehensively compared the succinic acid yield rate at different potentials. As shown in Fig. 3e, the NiO@PAA@NHS/Au@A. succinogenes biohybrid system reaches the highest succinic acid yield rate at −0.3 V (vs RHE), which can be attributed to a balance between electron supply and the metabolic demands of the bacteria. At −0.2 V (vs RHE), the photocurrent density is lower, suggesting that the electrons supply to the biohybrid system is insufficient to meet the bacteria’s needs for succinic acid production (Supplementary Fig. 18). This could be due to less efficient charge separation or a lower driving force for electron transfer at this potential. With increasingly negative potentials (−0.2 to −0.5 V vs RHE), the photocurrent density and H2 evolution on the bare NiO@PAA@NHS photocathode both rose markedly (Supplementary Fig. 19). However, the highest succinic acid yield occurred at −0.3 V, indicating that excessive bias promotes H2 generation rather than electron transfer to the bacteria for CO2 fixation49. These results suggest that hydrogen evolution and transmembrane electron uptake are competitive processes, and that −0.3 V represents an optimal potential where interfacial charge transfer and microbial metabolism are effectively balanced.

In addition to succinic acid, other byproducts such as acetic acid, formic acid, and pyruvic acid were also measured in the biohybrid system. As presented in Fig. 3f, the yield rate of succinic acid is the fastest, indicating that it is the predominant product under the given conditions, and it accounts for 57.18% of the total yield. To further analyze possible gaseous products, gas chromatography (GC) was performed to quantify H2 and CO generated during the reaction. As shown in Supplementary Fig. 20, trace amounts of CO (~425.7 nmol after 3 h) and small amounts of H2 (~144 μmol after 8 h) were detected. The calculated Faradaic efficiencies were 48.3% for the hydrogen evolution reaction (HER) and only 0.38% for CO formation from CO2 reduction, confirming that gaseous CO2RR products are negligible and that the majority of photogenerated electrons are utilized for microbial succinate synthesis rather than abiotic gas formation.

The long-term stability of the PEC-biohybrid system for succinic acid generation was evaluated to monitor the photocurrent (Supplementary Fig. 18) and cyclic PEC reactions (Supplementary Fig. 21). The photocurrent changes were negligible when the biohybrid system was operated at −0.3 V vs RHE over a period of 10 h, indicating a stable transfer of photoelectrons from the electrode to the bacteria. In addition, succinic acid yield was assessed over five consecutive cyclic reactions. There is a gradual decrease in succinic acid production after 5 days, which can be attributed to minor degradation of both the electrode materials and bacterial viability under prolonged PEC conditions.

Maintaining a consistent output is essential for the viability and efficiency of industrial production processes. Therefore, the continuous production of succinic acid by a biohybrid system under conditions that simulate day and night cycles, alternating between PEC and EC modes, was investigated. As shown in Fig. 3g, the simulation of a light–dark cycle of 12 h effectively mirrors the biohybrid system’s capability to produce succinic acid for 5 days, highlighting the system’s potential for sustained industrial application. It should be noted that the “superior efficiency” claim refers specifically to the higher succinic acid productivity achieved under PEC conditions (during the light phase), whereas EC conditions alone exhibit a relatively lower production rate. Nevertheless, alternating between PEC and EC modes is crucial to maintaining continuous and stable succinic acid production over extended periods.

Furthermore, to evaluate the biocompatibility of the biohybrid system, confocal laser scanning microscope (CLSM) images were taken before and after the PEC reaction (Supplementary Fig. 22). A. succinogenes was stained with the BB cell Probe N01™/PI fluorescent probe. The BB cell Probe N01™ can emit bright green fluorescence upon excitation due to its ability to penetrate the cell membrane of living bacteria. In contrast, PI can only stain dead bacteria, showing red fluorescence after excitation50–53. Compared to A. succinogenes before the reaction, after an extended reaction period, there was a significant increase in green fluorescence intensity and a slight enhancement in the red fluorescence signal. This suggests that the bacteria were able to rapidly multiply within the system and that there was a natural generational turnover occurring. Additionally, a colony-forming unit (CFU) assay was conducted to assess bacterial viability (Supplementary Fig. 23). There was an increase in the bacterial count after the long-term reaction. Collectively, these findings further suggest that the A. succinogenes in the biohybrid system possesses robust self-repairing and self-replicating capabilities, indicating that the bacteria remain viable and active before and after the reaction, which is crucial for sustained functionality in industrial applications.

The succinic acid yield rates in a PEC-biohybrid system were compared with various other biohybrid systems and bacterial strains under different reaction conditions (Supplementary Table 1). As shown in Fig. 3h, the biohybrid system operating in PEC mode demonstrated the highest succinic acid production rate among the systems tested, which is a testament to its superior efficiency. The PEC-biohybrid system’s superior succinic acid yield rate, high biocompatibility, and long-term stability position it as a leading candidate for industrial-scale production and commercialization of this essential chemical.

During succinic acid biosynthesis, synergistic catalysis between glucose and CO2 is essential. The carbon origin distribution in succinate was precisely mapped through comprehensive 1H and 13C nuclear magnetic resonance (NMR) spectroscopic analysis. In the 1H NMR spectrum (Supplementary Fig. 24a), the methylene proton peak appears at 2.34 ppm. Following 13CO2 labeling, this signal remains unchanged. However, when labeled with 13C6H12O6, the peak splits into a doublet, demonstrating that 13C from glucose is incorporated into the succinic acid carbon skeleton through metabolic processes, inducing complex coupling (13C-13C coupling). For the 13C-NMR analysis (Supplementary Fig. 24b), the carboxyl carbon (-COOH) resonates at 181 ppm, and its signal intensity increases significantly after 13CO2 labeling, confirming that CO2 fixation directly contributes to the carboxyl carbon. And the methylene carbon (-CH2-) appears at 32 ppm, and both the methylene and carboxyl carbon signals show notable enhancement upon 13C6H12O6 labeling. This indicates that the glucose carbon skeleton, via glycolysis/TCA cycle metabolism, generates the methylene group and partially contributes to the carboxyl carbons of succinic acid. This dual-labeling NMR analysis provides clear evidence for the distinct biosynthetic origins of succinic acid’s carbon backbone.

Mechanistic insights into enhanced succinic acid production
Figure 4a presents a schematic overview of succinic acid biosynthesis in the NiO@PAA@NHS/Au@A. succinogenes biohybrid, emphasizing glucose assimilation and redox modulation along the C4 metabolic pathway under photoelectrocatalytic conditions. In this system, electronic interactions at the biohybrid interface modulate intracellular redox states, thereby favoring succinate formation. Based on the additional electrochemical and control experiments, we suggest that direct electron transfer (DET) across the Au-mediated electrode-bacteria interface serves as the primary pathway, whereas H2-mediated electron transfer makes a minor contribution under conditions study. Cyclic voltammetry (CV) analysis (Supplementary Fig. 25a) revealed that wild-type A. succinogenes exhibited intrinsically weak electroactivity, while Au-adapted cells showed pronounced redox peaks centered at approximately +0.15 V (vs RHE). The current density nearly doubled from ~0.45 mA cm−2 to ~0.85 mA cm−2. In contrast, the CV profile of the cell-free supernatant was mostly featureless, ruling out contributions from soluble mediators or residual Au ions (Supplementary Fig. 25b). These results suggest that the Au nanoparticles formed near the inner membrane act as conductive nanoscale bridges, facilitating charge exchange between the photoelectrode and bacterial metabolic pathways without disrupting cellular viability. Despite the presence of these Au NPs, we have observed that the bacteria continue to divide (Supplementary Fig. 2). Moreover, the uniform distribution of Au NPs within each bacterium further substantiates the successful complexation of the Au NPs and their excellent biocompatibility. Furthermore, we compared the viability of A. succinogenes cultured directly in a high-concentration Au ion solution (2 μM) versus those cultured using a stepwise acclimation approach (0.5 μM, 1 μM, 1.5 μM, and 2 μM) with flow cytometry (Supplementary Fig. 3). As shown in Fig. 2f, the apoptotic analysis revealed that 73.1% of the 100,000 bacteria treated directly with 2 μM HAuCl4 underwent apoptosis. In contrast, when bacteria were adapted to 2 μM HAuCl4, the survival rates surged to 89.1%. This significant difference demonstrates that the ALE approach is an effective way to introduce high concentrations of Au NPs within bacterial cells, which is crucial for enhancing charge transfer efficiency. All these micromorphological and elemental evidence provide compelling evidence of the intracellular localization of Au NPs, highlighting their potential role in facilitating electron transfer processes within the bacteria and potentially enhancing the overall efficiency of the PEC-bacterial biohybrid system. The precise route of electron transport across the bacterial membrane, however, requires further elucidation. Although a minor indirect pathway may coexist, its contribution is small. Umol amounts of H2 generated at the photocathode surface can be oxidized by membrane-bound hydrogenases and introduced into the bacterial redox network; however, the addition of external H2 caused only a marginal increase in succinic acid production (Supplementary Fig. 26), confirming that the H2-mediated route contributes minimally compared with the dominant DET process.

Fluorescence lifetime measurements (Fig. 4b) showed that Au@A. succinogenes exhibited a lifetime of 1.89 ns, representing a 45% reduction relative to the control, demonstrating that the incorporation of Au nanoparticles substantially accelerates intracellular electron transfer and charge separation.

To gain a deeper understanding of the internal metabolism of A. succinogenes, the relative ATP level and NADH/NAD+ ratios were compared among three groups: A. succinogenes, NiO@PAA@NHS/A. succinogenes, and NiO@PAA@NHS/Au@A. succinogenes. As depicted in Fig. 4c, the ATP content in A. succinogenes increased by 21% upon biofilm formation with NiO@PAA@NHS, and this increase was even more pronounced, reaching 107%, in the NiO@PAA@NHS/Au@A. succinogenes system. This substantial boost in ATP is crucial for enhancing biochemical synthesis processes. Additionally, the NADH/NAD+ ratio showed a 32% increase in the NiO@PAA@NHS/A. succinogenes group relative to the pristine A. succinogenes, whereas it exhibited a 78% decrease in the NiO@PAA@NHS/Au@A. succinogenes group, as shown in Fig. 4d. This observed change is largely due to the conversion of NADH to NAD+ during the metabolic pathways leading to succinic acid production. Key enzymes in these pathways, including malate dehydrogenase (MDH), fumarase (FUM), and fumarate reductase (FRD), are involved in the oxidation of NADH to NAD+. Therefore, to direct the bacterial metabolism towards the C4 pathway for succinic acid synthesis, an increased production of NAD+ is necessary, which is facilitated by the PEC process.

To further unravel the mechanisms underlying succinic acid production in different systems, we analyzed the expression levels of key genes associated with mixed-acid synthesis using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) (Fig. 4e). Overall, upon attachment to the electrode, the expression of all genes in A. succinogenes was upregulated, indicating that efficient electron supply at the electrode modulates an increase in various metabolic gene products. This upregulation was observed in both the NiO@PAA@NHS/A. succinogenes and NiO@PAA@NHS/Au@A. succinogenes systems. PEPCK and PK are determinants of the C4 and C3 pathways, respectively, while FRD, PflB, and ACK correspond to the production of succinic acid, formic acid, and acetic acid, respectively. Despite the significant upregulation of PK, which is associated with pyruvate kinase, the concentration of pyruvate remained low (Supplementary Fig. 27a). Meanwhile, the product distribution analysis revealed a notable shift in metabolic selectivity upon system modification. For A. succinogenes, succinic acid accounted for merely 44% of total products, while integration of the electrode assembly elevated this proportion to 52% (Supplementary Fig. 27b). These findings indicate that the Au NPs coupled with the photoelectrode not only enhanced the succinic acid production kinetics but also improved its metabolic selectivity. Although the concentrations of acetic and formic acids increased, succinic acid remained the predominant product. This suggests that pyruvate in the system is primarily converted into oxaloacetate and then funneled through the C4 pathway to produce succinic acid.

Methods
Strain and media
Actinobacillus succinogenes 130Z (ATCC 55618) was obtained from Professor Wenming Zhang (Nanjing Tech University). The culture medium contained: 5 g/L yeast extract, 9.6 g/L NaH2PO4·2H2O, 15.5 g/L K2HPO4·3H2O, 10 g/L NaHCO3, 30 g/L glucose, 1 g/L NaCl, and 2.5 g/L corn steep liquor at 37°C and 170 rpm. The fermentation medium consisted of 10 g/L yeast extract, 1.36 g/L NaAc, 0.3 g/L Na2HPO4·12H2O, 1.6 g/L NaH2PO4·2H2O, 3 g/L K2HPO4, 1 g/L NaCl, 0.2 g/L MgCl2·6H2O, 7.5 g/L corn steep liquor, 0.2 g/L CaCl2 and 15 g/L Mg(OH)2CO3 for PH neutralizer. Glucose was added after sterilization at a concentration of 30 g/L for fermentation.

Preparation of NiO photocathode
NiO photocathodes were synthesized using a hydrothermal method. Firstly, nickel foam (Ni Foam) was cut into electrode sheets of 1 × 5 cm size, and sequentially cleaned with acetone, alcohol, and distilled water by ultrasonic cleaning for 15 min. The whole NF was directly immersed into a 3 M HCl aqueous solution at 90°C for 20 min in a static immersion to remove surface oxides. The NF was washed with distilled water, and the washed NF was dried under nitrogen. Next, Ni(NO3)2·6H2O (1 mmol) and urea (2 mmol) were dissolved in distilled water (15 mL) and stirred to form a green transparent solution. The pretreated NF (1 cm × 5 cm) was immersed into the solution in a Teflon reactor (25 mL) and kept at 100°C for 12 h. The electrodes were then removed, and the electrodes were placed in a water bath. After removing the electrodes, they were ultrasonically cleaned in ethanol and distilled water. Finally, the electrodes were calcined in a muffle furnace under air atmosphere at 250°C for 3 h.

Preparation of NiO@PAA@NHS photocathode
First, the NiONSs electrodes were immersed in an aqueous acrylic acid solution (30% w/w acrylic acid, 0.03% w/w N,N’-bis(acryloyloxy) cysteamine, and 0.15% w/w 2,2’-azobis(2-methylpropionamidine) dihydrochloride) for 2 h. Then, the immersed hydrogel was sealed and heated at 70°C for 30 min to form the PAA network, and finally dried under nitrogen flow. Subsequently, the prepared NiO@PAANSs electrode was placed in a mixture of 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (0.5% w/w) and N-hydroxylthiosuccinimide (0.25% w/w) aqueous solution and soaked for 5 min at room temperature, and then the hydrogel was further dried to obtain the NiO@PAA@NHS NSs photocathode.

Au-ALE of actinobacillus succinogenes
HAuCl4 solution was first added to 20 mL of culture solution to reach a concentration of 0.5 μM. After 12 h of incubation, the bacteria were inoculated into a new 20 mL culture solution, and HAuCl4 solution was added to make the concentration of HAuCl4 of 1.0 μM, 1.5 μM, and 2.0 μM to complete the ALE process. Bacterial cultivation was conducted for 12 h under each HClO4 concentration.

Construction of NiO@PAA@NHS/Au@A. succinogenes biohybrid
Different Au-ALE concentrations of Au@A. succinogenes were inoculated in 20 mL of culture broth, and HAuCl4 solution was added to make the concentrations 1 μM, 2 μM, 3 μM, and 4 μM, respectively. The bacteria were incubated anaerobically in a shaker at 37°C for 12 h. Subsequently, 10,000 g centrifugation was performed to collect the Au@A. succinogenes, and the culture broth was washed with 0.1 M phosphate buffered saline (PBS), washed three times to remove the medium, which was dispersed again in the fermentation broth, and the prepared NiO@PAA@NHS photocathode was placed in solution and incubated for 2 h to construct the NiO@PAA@NHS/Au@A. succinogenes hybrid photoelectrode.

Bacteria density on photoelectrode
High-resolution SEM images were acquired from five randomly selected fields of view on each electrode surface. The images were analyzed using ImageJ software, where bacterial cells were identified through threshold segmentation, followed by manual correction to ensure accuracy. The actual surface area of each field was calculated based on the scale bar, and bacterial counts were normalized accordingly to yield cells per mm2. The reported data represent the mean ± standard deviation of five independent regions.

Photoelectrochemical measurements
The photocatalytic test was completed with a three-electrode system; the reference electrode (RE) was Ag/AgCl, the counter electrode (CE) was a platinum electrode, and the working electrode (WE) was a biohybrid photoelectrode prepared as described in Construction of NiO@PAA@NHS/Au@A. succinogenes biohybrid, and the electrolyte was a bacterial fermentation broth. The reaction was carried out in a 100 mL reactor with a 50 mL working volume. The potential of the saturated Ag/AgCl was converted to that of the reversible hydrogen electrode (RHE) as follows in all the experiments:

ERHE (V) = EAg/AgCl + 0.059 × pH + 0.1976 (1)

CO2 was passed 30 min before the start of the reaction to remove dissolved oxygen. LSV and I–t curve are performed on an Autolab PGATAT302N (Metrohm, Switzerland) electrochemical workstation. For H2 supplementation controls, H2 gas was continuously supplied at 400 mL h−1 during operation. The PEC-biohybrid experiments were performed at an applied potential of −0.3 V versus RHE under illumination, whereas the biohybrid controls were operated in the dark under open-circuit conditions. All other experimental conditions were kept identical to those without external H2. Succinic acid production was quantified by HPLC.

Preparation of the thin-sectioned samples of the hybrid system
The cultured samples were harvested by centrifugation at 10,000 rpm for 10 min and washed three times with 0.1 M phosphate-buffered saline (PBS) to remove culture solution before further characterization. For sample preparation, the hybrid system was dissolved in 1 mL of PBS solution and then fixed using 4% glutaraldehyde at 4°C. After 2 h of fixation, the sample was washed twice with PBS solution at room temperature, then stained with fresh 1% OsO4 in PBS for 1 h. After washing two times with PBS, the sample was sequentially dehydrated in 50%, 70%, 85%, 95% (two times), and 100% (three times) acetone, each time lasting for 10–15 min. Part of the dehydrated sample was embedded and sequentially infiltrated with 1:2, 1:1, and 3:1 resin:acetone, followed by pure resin for three times. The sample was further baked at 70°C for 12 h for complete curing and cut into 70 nm thin sections with a diamond knife. Finally, the sample was deposited onto a Formvar coated copper grid and microscope slide for transmission electron microscopy and confocal fluorescent observation, respectively.

Biohybrid photoelectrode characterizations
Scanning electron microscopy (SEM) images were viewed with a Hitachi-S4800 scanning electron microscope. Transmission electron Microscope (TEM) images were viewed with a JEOL JEM-2100 scanning electron microscope. UV–vis absorption spectrum was recorded on an Evolution 789S spectrophotometer (Shanghai Light Technology Company). Confocal laser scanning microscopy (CLSM) images were obtained by a confocal laser scanning microscope (TCSSP8 MPS MD, LEICA, Germany). The light source was derived from a Xenon lamp (PLS-SXE300, Perfect Light, Beijing) with a filter (AM 1.5G, Perfect Light, Beijing).

Analytical methods
The concentrations of organic acids were determined using Shimadzu high-performance liquid chromatography (Japan). Separation of the samples was achieved using an Ion exclusion chromatography column (Shim-pack SCR-101H) under the following conditions: a column temperature of 55°C, a mobile phase of 100% 0.25 g/L H2SO4, and a flow rate of 0.5 mL/min. Succinic acid, pyruvic acid, formic acid, and acetic acid were detected were detected at 214 nm using an SPD-20A detector. An automatic sampler was used to inject 20 μL of the samples into the chromatograph.

Determination of CO2 conversion efficiency
CO2 flow rate was monitored continuously with a calibrated mass-flow meter and fixed at 7 mL min−1 (at 298 K and 1 atm). The molar CO2 feed rate was therefore:

n(CO2, input) = 7 mL min−1 / 24450 mL mol−1 × t (min) = 2.86 × 10−4 t mol (2)

Succinic acid was detected by HPLC; its molar yield was quantified at different reaction durations. The CO2 conversion efficiency was then calculated as:

CO2 Conversion Rate (%) = (Succinic Acid Yield (mol) / CO2 Input (mol)) × 100% (3)

Glucose concentration analysis
The glucose level was measured using the Beyotime Glucose Assay Kit with O-toluidine, which is based on the chromogenic reaction between glucose and o-toluidine, with quantification performed by colorimetric analysis.

Faradaic efficiency calculation
The Faradaic efficiencies (FEs) for gaseous products were calculated based on the total charge passed during PEC and the quantified number of products formed. The total charge Q (C) was obtained by integrating the photocurrent over the reaction time, and the molar amount of each product n (mol) was determined from gas chromatography (GC) or high-performance liquid chromatography (HPLC) analysis. The FE for each product was calculated using the following equation:

FE = (n × z × F) / Q × 100% (4)

Where n is the number of moles of the detected product, z is the number of electrons transferred per molecule (2 for both H2 and CO), and F is the Faraday constant (96485 C mol−1).

Cyclic voltammetry measurements
Cyclic voltammetry (CV) was performed in a three-electrode configuration using carbon paper (1 × 1 cm2) as the working electrode, a Pt foil/wire as the counter electrode, and a saturated Ag/AgCl electrode as the reference electrode. The electrolyte was 10 mM phosphate-buffered saline (PBS, pH 7), deaerated with CO2 for 30 min prior to measurements and maintained under a continuous CO2 atmosphere. All measurements were conducted at 37°C. Wild-type and Au@A. succinogenes cells were harvested by centrifugation, washed three times with PBS, and resuspended to OD600 ≈ 2.0. The carbon paper was immersed in the bacterial suspension and incubated anaerobically at 37°C for 4 h to allow cell attachment, then gently rinsed with PBS and used immediately. CV was recorded in the potential range of −0.4 V to 1.4 V (vs RHE) at scan rates of 5–50 mV s−1. Each voltammogram represents a stable response after three consecutive scans. Measurements were repeated with independently prepared bacterial suspensions (n=3). Cell-free supernatants were tested under identical conditions as controls.

NAD+/NADH assay kit measurements
NAD+/NADH assay kit (Beyotime Biotechnology) with WST-8 was used to determine the concentration of NADH and NAD+ in A. succinogenes following the standard manual of the assay kit. All experiments were conducted in the same fermentation broth under identical anaerobic conditions; control and biohybrid samples were cultured for the same duration, with the latter operated under light illumination and applied potential.

Measurement of ATP content
First, 200 μL lysate was gently added to promote cell lysis, and centrifugation was performed to obtain the solution. The solution was added to the 96-well plate, and placed at room temperature for 3 min. After adding 100 μL sample, the RLU value was determined by luminometer.

A. succinogenes viability
During the long-term test, the viability of A. succinogenes has been determined by colony-forming units (CFU) assays. At fixed time intervals, aliquots of 500 μL reaction solution were collected by a sealing syringe. 100 μL of the collected sample was serially diluted with saline. 100 μL of the original and diluted samples were spread on sterilized nutrient agar plates, then incubated at 37°C for 12 h. Finally, the survival number of cells (in CFU) was quantified by counting the visible colonies and the survival rate was calculated.

Staining analysis of bacteria
The bacteria before and after reactions were resuspended in 100 μL BB cell Probe N01 Dye solution A and PI Dye solution B. After incubating at room temperature for 15 min, the bacteria were washed with 0.85% NaCl solution once and then suspended again. Finally, add 20 μL bacterial to the confocal surface dish, and the samples were imaged with CLSM under the excited laser of 488 nm, the emission wavelength of 525 and 620 nm, respectively.

Flow cytometry analysis
A. succinogenes cells were harvested, washed, and resuspended in phosphate-buffered saline (PBS, pH 7.4) prior to measurement. All samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) equipped with 488 nm and 638 nm lasers. Forward scatter (FSC) and side scatter (SSC) were used to gate bacterial populations based on size and granularity, and a threshold was set on SSC to exclude debris and electronic noise. A minimum of 50,000 events per sample were recorded within the bacterial gate. Data acquisition was performed using CytExpert software (version 2.4, Beckman Coulter), and all collected flow cytometry standard (FCS) files were subsequently analyzed with FlowJo software (version 10.8, BD Biosciences, San Jose, CA, USA). The analysis included gating on singlets, exclusion of outliers, and quantification of fluorescence-positive subpopulations based on unstained and single-stained controls. All experiments were performed in at least three independent biological replicates, and representative histograms or dot plots are shown.

Quantitative RT-PCR
Total RNA Extractor (Trizol) was used to extract total RNA. Real-time quantitative PCR technology refers to the experimental method of quantitative analysis of the starting template by adding a fluorescent group (SYBR green I) to the PCR reaction system, and using the accumulation of fluorescent signals to monitor the PCR process in real time. The 16S rRNA gene was used to standardize the mRNA levels. Primer sequences for qRT-PCR are indicated in Supplementary Table 3. The mRNA level of the target genes was calculated via the 2−ΔΔCT method.

Statistics
Statistical analysis was performed using GraphPad Prism 8.0.2 software using a two-tailed t-test analysis of variance hypothesis. Significant differences are marked as *P < 0.05, **P < 0.01, and ***P < 0.001. All data are presented as mean ± SD. The number of biologically independent samples for each panel was three unless otherwise stated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Authors constructed a PEC-bacterial biohybrid system to produce succinic acid in a sustainable method compared to the current. They obtained a very high yield rate and other important conclusions. Before its publication, the following questions should be addressed.\n1. In Fig.1, bacteria were adsorbed onto the photoelectrode surface. Have bacteria desorbed from the surface?\n2. In Fig.2c,I just noticed black particles located inside bacteria not outside bacteria. Thus did that (no particles outside bacteria) affect electrons transfer from outside to inside?\n3. In the manuscript, authors stated \"the Au ions are reduced to Au NPs by reductive substances present in the bacterial cells, such as NADH or other biomolecules with reducing properties \". I just wonder what kind of enzymes were responsible for this reduction?\n4. There were a lot of by products,leading to a relatively low selectivity of succinic acid (<60%) how to handle them? Did authors make a discussion on this phenomenon？\n5. Actinobacillus succinogenes may not belong to electroactive microorganism. In Fig.4a, authors provided a mechanism draft and pointed out that \"This internal conductive network not only allows for electron transfer through cytochromes Cyt d I and Cyt d II but also plays a role in the synthesis of additional redox couples\". Here, I am still wondering how electrons could transfer from electrode into intracellular? Please provide more information for cytochromes Cyt d I and Cyt d II to prove their functions of electrons transfer.\n6. In Fig.3g, 48 h seemed to be a little short to support the long term experimental results and the stability of the biohybrid system.", "reviewer_2": "This work describes the assembly of A. succinogenes onto photoelectrodes containing a hydrogel, where the authors claim that the hydrogel forms covalent bonds with dangling amines in the cell membrane-bound cytochromes. The authors use this photoelectrochemical hybrid system to generate succinic acid using CO₂ and glucose as carbon sources. They assert that their system outperforms other succinic acid-producing biohybrids and propose a tentative mechanism suggesting that extracellular electron transfer plays a critical role. However, they do not provide experimental evidence to support this proposed mechanism.\nThe data presentation is generally adequate, although in several instances, additional information would enhance clarity and experimental reproducibility, both in the form of plotted data and methodological details. For example, when comparing succinic acid formation rates in photoelectrochemical biohybrids with electrochemical and photochemical biohybrids, no supporting data—such as chronoamperometry, faradaic efficiency, external quantum yield, or details on the methods used for these experiments—is provided. Additionally, in some plots, it is unclear whether the data were obtained from triplicate experiments.\nOverall, while the manuscript presents a concept (covalent linkage of bacteria to a (photo)electrode) that is interesting and noteworthy for the field of biohybrids, the described results from this system provide only an incremental advancement compared to the state-of-the-art literature. Although the figures are well presented, the manuscript would greatly benefit from more exhaustive supporting information to substantiate its claims and a more detailed methods section. Furthermore, throughout the manuscript the authors frequently overstate their claims without sufficient supporting evidence, with the conclusions also included. For these reasons, I believe this manuscript does not meet the standards of Nature Communications. A more specialized journal would be a better fit, particularly after addressing the points raised above.\nRegardless of this, I have some questions and suggestions for the authors to address, that I hope will help them to improve their manuscript:\n• Abstract: If space allows, including more details on system metrics such as photocurrent, faradaic efficiency, applied potential, and CO₂ conversion efficiency to succinate would strengthen the abstract.\n• Terminology: The authors use the terms \"domestication\" and \"acclimated\" when describing microbial exposure to gold nanoparticles. Are they referring to adaptive laboratory evolution (ALE)? If so, I recommend replacing these terms with \"adaptation\", \"adapted\", and \"ALE\" for consistency with literature. Additionally, relevant references on ALE, such as Peidong Yang’s work on methanol adaptation of S. ovata, should be cited.\n• Gold Nanoparticles (AuNP) & Microbial Exposure:\no How long were the cells exposed to the presence of HAuCl₄ during sequential exposure to 1, 1.5, and 2 µM HAuCl₄ solutions?\no What was the size of the synthesized AuNP? Do the authors believe that plasmonic effects could play a role in this Au-containing biohybrid system?\no The authors claim that AuNP accumulate near the cell membrane, but Figure 2e appears to show AuNP accumulation mainly in the cytoplasm rather than at the membrane. Additionally, this figure is difficult to interpret due to its size; scaling it up could improve clarity.\n• Hydrogel Layer & Bacterial Attachment:\no What is the porosity of the hydrogel layer? Can bacteria go inside any pores?\no How do the authors confirm that NHS has reacted with the amine groups on the bacterial surface? This would help support their proposed hypothesis and mechanism, later on.\no The authors claim that A. succinogenes contains dangling NH₂ groups in its membrane-bound cytochromes (Cyt d I and d II), allowing covalent attachment to the hydrogel. However, Ref 40 does not provide any information on amines in membrane-bound cytochromes. Could the authors provide supporting data to their claim and cite appropriate literature references?\no The manuscript frequently refers to \"bacterial adhesion\" and \"surface functionalization\" as key factors in improving attachment and not covalent linkage. However, Figure 1 depicts the bacteria as covalently linked to the hydrogel, yet no experimental evidence confirms this covalent bonding (bacteria-NH-CO-hydrogel). Unless further characterization proves covalent attachment, I strongly recommend the authors tone down their claims and modify Figure 1 (and any related figures) accordingly.\no Cell Density in Figure 2j: I could not find details in the methods section regarding how the authors quantified the number of cells per mm². This information should be included.\n• Photoelectrochemical (PEC) Experiments:\no What are the faradaic efficiency of all products (coming from the (photo)electrodes and bacteria) and CO₂ conversion efficiency to succinate?\no Were there any byproducts, such as H₂ or CO, formed at the photocathodes?\no The authors show that microbes can produce succinate without CO₂. If PBS medium was used as the electrolyte, was glucose also present as a carbon source in the PEC experiments?\no The manuscript should explicitly state the carbon source used when Ar is present, and the methods section should clarify the media and conditions used for PEC experiments.\no The manuscript lacks experimental data and explanations for electrochemical biohybrids (EC) and photochemical biohybrids (PC) (Figure 3b).\no Regarding Figure 3b, the text only mentions the succinate formation rate, but without additional data, it is unclear why the EC-biohybrid system performs worse than the PEC-biohybrid system.\no Biohybrid experiments typically run for dozens of hours or days, yet the PEC chronoamperometry experiments presented here last only 2 hours. How long were the biohybrid experiments run for Figures 3b, 3c, and 3e?\no In Supplementary Figure 14, how long is each cycle? If it is 10 hours per cycle, this should be explicitly stated for clarity.\no Figure 3g and Supplementary Figure 14 lack error bars—were these plots generated from triplicate experiments?\n• Electron Transfer Pathway & Proposed Mechanism:\no Figure 4 clearly depicts the metabolic pathway for microbial CO₂ and glucose conversion to succinic acid. However, the extracellular electron transfer pathway remains unclear. No data (e.g., spectroscopy results) support the hypothesized extracellular electron transfer mechanism. One suggestion is to use time-resolved absorption or vibrational spectroscopy to determine whether the AuNP are reduced/oxidized by internal microbial electron transport chains.\no Based on Ref 40, A. succinogenes contains membrane-bound cytochromes and hydrogenases. Given the (photo)currents at -0.3 V vs. RHE, it is likely that the microbes are not able to uptake all charges and use instead in situ-produced H₂ from the (photo)electrodes as an electron donor for succinic acid production. Therefore, a key control experiment to test this point would be using a knockout strain lacking membrane-bound hydrogenases to see whether succinic acid production is affected under the same conditions.\no To confirm that succinic acid is derived from CO₂ (and not solely from glucose), ¹³CO₂ and ¹³C-glucose labelling experiments should be conducted.\n• References:\no References 30 and 31 are incorrect—please verify them and check the rest.", "reviewer_3": "The authors demonstrate a certain range and novelty compared to the current state of the art. However, I must state that the manuscript, in its current form, does not meet the journal's standards. Please see the comments below for the reasons behind this. Nevertheless, I see potential for the manuscript to meet these standards, provided the comments are addressed.\nMajor points:\nThe text describes the evaluation of linear sweep voltammetry between Ar- or CO₂-saturated electrolyte under the influence of light or in darkness. Consequently, a “significantly enhanced” current density is reported both in the light and in the dark under a CO₂ atmosphere. However, two points need to be critically examined: Firstly, the difference between the two experiments conducted in darkness is relatively small. Secondly, the results presented appear to be based on a single measurement (n = 1), which does not allow for any statistically reliable conclusion to be drawn about the observed differences. This should be clearly stated in the discussion. Additionally, the statement on succinate yields under CO₂ or Ar conditions is redundant. It is well known that succinate production in A. succinogenes is CO₂-dependent via the reductive branches of the TCA cycle (see work by James B. McKinlay).\nIn general, the manuscript and the figures lack precise information on the statistics, particularly regarding the sample size. For instance, some diagrams in Fig. 3/4 display a standard deviation, while others do not. There is no explanation for this either in the captions or in the text. It also remains unclear whether the data are technical or biological replicates. Without this information, it is difficult to assess the reliability of the results, which significantly hinders interpretation, especially as the text frequently refers to “significant” changes. This needs to be clarified.\nRegarding the results in Fig. 3g, the data shown correspond to a light-dark cycle intended to simulate a day-night rhythm. The simulations indicate low productivity during the night phases. This implies that the improvement in succinate production rates (g/L/h) emphasised in this manuscript actually occurs only during the day phase. Over a multi-day fermentation process, the average productivity would therefore be lower, calling into question the claimed “superior efficiency” of the system. This limitation should be acknowledged in the discussion.\nIn general, it would be of interest to analyse the results over a complete fermentation run rather than solely the initial hours. In addition to concentration data, the yield in terms of metabolised glucose (g/g) should also be considered. This would indicate whether not only the production rate of succinate (g/L/h) but also the overall yield (g/g) is improved. In other words, it would determine whether more carbon is actually converted into the target product, succinate. This additional consideration would be crucial in fully evaluating the efficiency of the process.\nThe manuscript lacks a critical discussion of the challenges posed by the use of light in large-scale applications, despite the method being lauded in the abstract. Typical stirred tank reactors for industrial applications would either require integrated light sources or be replaced by alternative, scalable reactor systems capable of supporting both the electrochemical (electrodes) and photochemical components. At present, the scale-up of bioelectrochemical systems remains a significant challenge. Moreover, research on phototrophic organisms, such as cyanobacteria, has demonstrated that incorporating light into biotechnological processes presents considerable obstacles, including limited light penetration in the reactor (restricting reactor diameter), shading issues, and more. These challenges should be addressed in the discussion and conclusion, especially since the system is described as a “leading candidate for large-scale production and commercialisation.”\nThe section from “Upon the absorption of the substrate glucose by the bacteria, it is converted into phosphoenolpyruvate (PEP), marking the beginning of the metabolic divergence into two distinct pathways.” to the end of the paragraph serves as a general summary of the metabolism of A. succinogenes. This content is more appropriate for the introduction rather than the results or discussion sections. Furthermore, this section lacks references to substantiate the metabolic processes described. Appropriate sources must be included.\nGene expression analysis of key metabolic enzymes shows an upregulation of all studied enzymes compared to the control, including the enzymes PK, ACK, and PfIB, which are involved in by-product reactions. Consequently, it would be important to compare the yields of by-products between the control and the biohybrids. The text merely states: “Although the concentrations of acetic and formic acids increased,” yet concrete data are missing. By what proportion did the by-products increase? Was there a shift in the ratio between by-products and main products? These aspects are not discussed further, despite the upregulation of the relevant enzymes. This section should be expanded upon, and the corresponding data on by-products should be included.\nMinor points:\nThe manuscript lacks headings and subheadings to provide a clearer structure.\nThe division of individual figures into up to ten sub-figures, each employing very different methods (e.g., diagrams, microscopic images from different microscopes), makes the manuscript difficult to follow and diminishes readability. To enhance clarity, these illustrations should be divided into multiple smaller figures and presented sequentially within the text."}, "author_response": "Response to the reviewers’ comments for Manuscript (number: NCOMMS- 25-14204-T)\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nAuthors constructed a PEC-bacterial biohybrid system to produce succinic acid in a\nsustainable method compared to the current. They obtained a very high yield rate and\nother important conclusions. Before its publication, the following questions should be\naddressed.\nResponse: We sincerely appreciate the reviewer’s positive assessment of our work and\ntheir constructive feedback. We have carefully addressed all the raised points in our\nrevisions and provided detailed responses to each comment. We believe these revisions\nnow align with the reviewer’s rigorous standards and significantly strengthen the\nmanuscript. Thank you for your valuable guidance throughout this process.\n1. In Fig. 1, bacteria were adsorbed onto the photoelectrode surface. Have bacteria\ndesorbed from the surface?\nResponse: We really appreciate the reviewer’s valuable comment. To assess the binding\nstability of A. succinogenes on the photoelectrode, we conducted confocal laser\nscanning microscope (CLSM) imaging of NiO@PAA@NHS/Au@A. succinogenes\nphotoelectrode after immersion in PBS solution for 30 and 60 minutes in PEC\nconditions. The bacteria were stained with N01 fluorescent dye.\nFig. R1-1 CLSM images of A. succinogenes dyed with BBcellProbe N01 after immersing in PBS\nsolution for (a) 30 min and (b) 60 min.\nAs shown in Fig. R1-1, the minimal change in fluorescence intensity indicates\nnegligible bacterial detachment. These findings, along with FTIR evidence of covalent\namide bond formation between NHS groups and bacterial amines (Fig. R1-2), confirm\nthe robust and stable immobilization essential for long-term PEC performance.\nFig. R1-2 Fourier transform infrared spectroscopy (FTIR) of NiO@PAA@NHS/A. succinogenes\nand A. succinogenes.\n2. In Fig. 2c, I just noticed black particles located inside bacteria not outside bacteria.\nThus, did that (no particles outside bacteria) affect electrons transfer from outside to\ninside?\nResponse: We sincerely thank the reviewer for this insightful observation. It is indeed\ncorrect that the Au nanoparticles (Au NPs) are primarily localized intracellularly,\nespecially near the inner membrane, as confirmed by TEM (Fig. 2c), HAADF-STEM\nimaging (Fig. 2d), and EDS elemental mapping (Fig. 2e). This spatial distribution is a\nresult of the adaptive laboratory evolution (ALE) process, where A. succinogenes were\ngradually acclimated to increasing concentrations of HAuCl , enabling them to reduce\ndependent reductases. This controlled intracellular accumulation is essential to\nforming a conductive nanobridge that facilitates internal redox reactions without\ncompromising cell viability.\nDespite the lack of extracellular Au NPs, the electron transfer from the external\nphotoelectrode to the bacteria is not hindered. On the contrary, our design enables a\nsynergistic extracellular-intracellular electron transfer pathway: At the interface, the\nNiO@PAA@NHS photoelectrode forms amide bonds with the bacterial surface via\nNHS ester groups reacting with membrane-exposed amines, as confirmed by FTIR (Fig.\nR1-2). This creates a stable and close-contact interface facilitating efficient electron\ninjection from the photoelectrode. Within the bacteria, the Au NPs near the membrane\nact as electron mediators, accepting electrons that traverse the bacterial envelope and\nrelaying them deeper into intracellular metabolic pathways.\nFig. R1-3 Fluorescence spectra of A. succinogenes before and after PEC reactions (A.\nsuccinogenes@Au in biohybrid was stained by DiOC (3) green membrane potential dyeing probe\n2\nin advance, λexc = 482 nm).\nTo further verify the successful transmembrane electron transport, we conducted\nfluorescence spectroscopy experiments using DiOC (3) staining. DiOC (3) is a\n2 2\nmembrane-potential-responsive fluorescent probe that binds to bacterial membranes\nand emits green fluorescence. When the membrane potential changes due to external\nstimuli, the dye molecules undergo self-polymerization, causing the fluorescence to\nredshift. As shown in Fig. R1-3, a redshift in fluorescence post-PEC is presented,\nindicating electron accumulation across the bacterial membrane.\nThus, although the Au NPs are intracellular, their strategic localization near the\ninner membrane ensures they serve as a conduit between extracellular electrons and\nintracellular metabolism. This architecture significantly boosts succinic acid\nproduction by enabling A. succinogenes to efficiently harness photoelectrons.\nWe hope this expanded explanation clarifies that the observed Au NP localization not\nonly supports but also enhances the electron transfer process and is a key feature of\nour biohybrid design.\n3. In the manuscript, authors stated \"the Au ions are reduced to Au NPs by reductive\nsubstances present in the bacterial cells, such as NADH or other biomolecules with\nreducing properties \". I just wonder what kind of enzymes were responsible for this\nreduction?\nResponse: Thank you very much for this insightful question. The intracellular reduction\nof Au3+ to elemental Au0 is indeed a critical step in the formation of biohybrid\nnanostructures and is primarily mediated by NADH-dependent reductase enzymes.\nSeveral studies have identified key enzymes involved in such metal ion reductions: (1)\nCorrea-Llantén et al. demonstrated that the thermophilic bacterium Geobacillus sp.\nstrain ID17, isolated from Deception Island, Antarctica, can intracellularly reduce Au3+\nto elemental gold (Au) using NADH as a cofactor, with the reduction process mediated\nby enzymes (Microbial Cell Factories 2013, 12:75); (2) Dhanasekar et al. reports that\nthe fungus Bipolaris oryzae produces NADPH-dependent reductase in its cell-free\nextract, which rapidly reduces Au3+ ions to Au0, resulting in the formation of gold\nnanoparticles within 5–10 minutes (ChemistrySelect 2020, 5, 11522– 11529).\nAlthough A. succinogenes has not yet been reported for specific Au-reductase\nenzymes, genome annotations (via KEGG and UniProt) reveal the presence of multiple\nNAD(P)H-dependent oxidoreductases, including ferredoxin-NADP⁺ reductase and\ndihydrolipoamide dehydrogenase, both of which have redox potentials compatible with\nAu3+ reduction. As for mechanism of the enzyme, the NADH-dependent reductase\npresumably mediates electron transfer from NADH to Au3+, reducing it to metallic gold.\nThis enzyme may function in coordination with other intracellular enzymes to complete\nthe reduction process of Au ions.\nFurthermore, the intracellular localization of Au NPs near the inner membrane\n(Fig. 2c–e) and the successful adaptive laboratory evolution (ALE)-mediated tolerance\nof Au3+ toxicity imply the active involvement of membrane-associated reductase\nenzymes during stepwise adaptation. These enzymes may function synergistically with\nintracellular electron donors (e.g., NADH, glutathione) to nucleate and stabilize Au\nNPs. We thank the reviewer again for highlighting this important mechanistic point.\n4. There were a lot of by products, leading to a relatively low selectivity of succinic\nacid (<60%) how to handle them? Did authors make a discussion on this phenomenon?\nResponse: Thank you for this valuable question regarding product selectivity, which is\nindeed crucial for the economic and environmental feasibility of biohybrid-based\nsuccinic acid production. In our system, the main by-products detected include formic\nacid, acetic acid, and pyruvic acid, which are typical intermediates of the mixed-acid\nfermentation pathway employed by Actinobacillus succinogenes. The metabolic\nnetwork of A. succinogenes naturally branches between the C3 pathway (leading to\nacetic/formic acid via pyruvate and acetyl-CoA) and the C4 reductive TCA pathway\n(leading to succinate), which compete for common precursors and cofactors such as\nNADH and PEP (phosphoenolpyruvate).\nImportantly, our PEC-biohybrid system increased the succinic acid selectivity\nfrom 44% (in native A. succinogenes) to 52% (with the NiO@PAA@NHS/Au@A.\nsuccinogenes photoelectrode), as shown in Fig. R1-4.\nFig. R1-4 Product distribution ratio diagram after 12 hours of PEC (NiO@PAA@NHS/A.\nsuccinogenes and NiO@PAA@NHS/Au@A. succinogenes) reaction and PC reaction (A.\nsuccinogenes).\nThis improvement is attributed to several synergistic factors: (1) Photogenerated\nelectrons preferentially support the reductive C4 pathway by enhancing the\nintracellular NAD⁺/NADH balance (Fig. 4e), thereby favoring fumarate reductase\n(FRD) activity, which catalyzes the final step of succinate production; (2) Upregulation\nof PEPCK and FRD gene expression under PEC conditions (Fig. 4f) indicates a\nmetabolic shift toward the C4 pathway; (3) The internal Au NPs facilitate enhanced\nelectron flux across the bacterial membrane, reducing redox bottlenecks and directing\nmetabolic flux toward succinate. Nonetheless, we fully acknowledge that achieving\nselectivity >60% remains a challenge. In future work, product specificity can be\nenhanced through metabolic engineering of A. succinogenes, photoelectrode and\nelectron supply optimization, and post-reaction purification and separation.\n5. Actinobacillus succinogenes may not belong to electroactive microorganism. In Fig.\n4a, authors provided a mechanism draft and pointed out that \"This internal conductive\nnetwork not only allows for electron transfer through cytochromes Cyt d I and Cyt d II\nbut also plays a role in the synthesis of additional redox couples\". Here, I am still\nwondering how electrons could transfer from electrode into intracellular? Please\nprovide more information for cytochromes Cyt d I and Cyt d II to prove their functions\nof electrons transfer.\nResponse: We sincerely appreciate the reviewer’s critical question, which brings\nattention to the essential mechanism of electron uptake in our PEC-bacterial hybrid\nsystem. It is correct that Actinobacillus succinogenes is not considered a classical\nelectroactive microorganism. However, our system was specifically engineered to\novercome this limitation by establishing artificial electron transfer pathways using a\ncombination of (i) NiO@PAA@NHS photoelectrodes, (ii) NHS-mediated covalent cell-\nelectrode binding, and (iii) intracellular Au nanoparticle (AuNP) networks that act as\nintracellular charge mediators.\nCytochrome bd oxidases (Cyt b I and Cyt b II are terminal oxidases that facilitate\nelectron transfer from the ubiquinol pool to molecular oxygen. These proteins are also\nannotated in the A. succinogenes genome and classified under EC 7.1.1.7. Structurally,\nthese complexes contain heme b , heme b , and heme d, which serve as redox centers.\n550 595\nStudy by Safarian et al. (Science 352. 583–586 (2016)) demonstrated that these\nenzymes form multi-heme chains capable of mediating long-distance electron transfer\nacross the membrane. Importantly, they contain exposed lysine and arginine residues,\nwhich provide primary amine groups that can covalently bind with NHS esters on the\nhydrogel, as supported by FTIR shifts in amide bond vibrations (Fig. R1-2). Thus, in\nour design, these cytochromes serve dual roles: (1) Electron acceptors at the cell-\nelectrode interface, receiving electrons from the NiO electrode via the PAA@NHS\nhydrogel; (2) Redox conduits into the intracellular quinone pool, linking external\nphotogenerated electrons to internal energy metabolism.\nTo support this hypothesis, membrane potential shifts were observed in the PEC\nsystem (Fig. R1-3b), confirming transmembrane electron transport; time-resolved\nfluorescence lifetime measurements showed a ~45% reduction in lifetime for Au@A.\nsuccinogenes, consistent with faster photoexcited carrier relaxation and effective\ncharge transfer (Fig. R1-5).\nFig. R1-5 Fluorescence lifetime spectra in A. succinogenes and Au@A. succinogenes.\nWe hope this detailed explanation clarifies how cytochrome bd oxidases contribute\nto the artificial electron uptake mechanism in our PEC-biohybrid system, and we thank\nthe reviewer again for prompting this deeper discussion.\n6. In Fig. 3g, 48 h seemed to be a little short to support the long-term experimental\nresults and the stability of the biohybrid system.\nResponse: We agree the previously shown 48 h experiment was insufficient. In the\nrevised version (Fig. R1-6), we provide data from a 5-day continuous operation,\nshowing sustained succinate production and system stability under alternating PEC/EC\nconditions. While minor performance decline was observed, the system maintains\nfunctionality, indicating its viability for longer-term applications.\nFig. R1-6 Long-term (5 days) succinic acid generation on NiO@PAA@NHS/Au@A. succinogenes\nbiohybrid in alternating PEC and EC models at -0.3 V vs RHE.\nReviewer #2 (Remarks to the Author):\nThis work describes the assembly of A. succinogenes onto photoelectrodes containing\na hydrogel, where the authors claim that the hydrogel forms covalent bonds with\ndangling amines in the cell membrane-bound cytochromes. The authors use this\nphotoelectrochemical hybrid system to generate succinic acid using CO and glucose\n2\nas carbon sources. They assert that their system outperforms other succinic acid-\nproducing biohybrids and propose a tentative mechanism suggesting that extracellular\nelectron transfer plays a critical role. However, they do not provide experimental\nevidence to support this proposed mechanism. The data presentation is generally\nadequate, although in several instances, additional information would enhance clarity\nand experimental reproducibility, both in the form of plotted data and methodological\ndetails. For example, when comparing succinic acid formation rates in\nphotoelectrochemical biohybrids with electrochemical and photochemical biohybrids,\nno supporting data—such as chronoamperometry, faradaic efficiency, external quantum\nyield, or details on the methods used for these experiments—is provided. Additionally,\nin some plots, it is unclear whether the data were obtained from triplicate experiments.\nOverall, while the manuscript presents a concept (covalent linkage of bacteria to a\n(photo)electrode) that is interesting and noteworthy for the field of biohybrids, the\ndescribed results from this system provide only an incremental advancement compared\nto the state-of-the-art literature. Although the figures are well presented, the manuscript\nwould greatly benefit from more exhaustive supporting information to substantiate its\nclaims and a more detailed methods section. Furthermore, throughout the manuscript\nthe authors frequently overstate their claims without sufficient supporting evidence,\nwith the conclusions also included. For these reasons, I believe this manuscript does\nnot meet the standards of Nature Communications. A more specialized journal would\nbe a better fit, particularly after addressing the points raised above. Regardless of this,\nI have some questions and suggestions for the authors to address, that I hope will help\nthem to improve their manuscript:\nResponse: We sincerely thank the reviewer for their thoughtful and constructive\nevaluation of our manuscript. We highly appreciate your recognition of the conceptual\ninterest in our work, particularly the development of a covalently bound\nphotoelectrochemical (PEC) biohybrid system using Actinobacillus succinogenes for\nsustainable succinic acid production. In response to your critical suggestions, we have\ncarefully revised the manuscript throughout. We have provided additional experimental\nevidence to support our proposed mechanism of extracellular-to-intracellular electron\ntransfer, including fluorescence lifetime spectroscopy and membrane potential analysis.\nIn addition, we have included detailed descriptions of control experiments, such as\nelectrochemical (EC) and photocatalytic (PC) biohybrids, along with\nchronoamperometry data and reaction durations. We clarified data reproducibility\nacross all figures and explicitly stated the number of replicates (n = 3) in captions, and\nexpanded the Methods section with precise experimental protocols to enhance\nreproducibility.\nWe genuinely appreciate your critical perspective, which has helped us\nsubstantially strengthen the scientific rigor, clarity, and transparency of our manuscript.\nWe hope that the revised version now better reflects the novelty and significance of our\ncontribution to the field of biohybrid systems. Thank you again for your valuable\nfeedback.\n• Abstract: If space allows, including more details on system metrics such as\nphotocurrent, faradaic efficiency, applied potential, and CO conversion efficiency to\n2\nsuccinate would strengthen the abstract.\nResponse: We appreciated the reviewer’s valuable suggestion. We have revised the\nabstract to include key system metrics: photocurrent density (1.9 mA cm-2), CO\n2\nconversion efficiency (67%), glucose conversion (0.78 g/g), and applied potential (-0.3\nV vs. RHE). These additions strengthen the practical and technical framing of our study.\nWe did not provide Faraday efficiency value in this work. The primary reason for not\nproviding Faradaic efficiency data is that, unlike traditional inorganic electrocatalytic\nsystems where electron transfer pathways and products are well-defined, the\nphotoelectrochemical-biohybrid system presents inherent complexities. In such hybrid\narchitectures, microorganisms not only serve as biocatalysts but also require energy\nallocation for self-maintenance. Thousands of concurrent biochemical reactions\ninvolving electron participation occur simultaneously, complicating the precise tracing\nof electron flux. Furthermore, the energy supply in this system is not exclusively derived\nfrom photoelectrode-generated electrons, which primarily function as auxiliary\ncontributors. A significant portion of metabolic energy originates from the organism's\ninternal ATP conversion processes. Consequently, the conventional concept of\nFaradaic efficiency becomes less critical and less applicable in evaluating the\nperformance of biohybrid systems, as it fails to account for these multifaceted energy\ntransduction mechanisms and distributed electron utilization pathways.\n• Terminology: The authors use the terms \"domestication\" and \"acclimated\" when\ndescribing microbial exposure to gold nanoparticles. Are they referring to adaptive\nlaboratory evolution (ALE)? If so, I recommend replacing these terms with\n\"adaptation\", \"adapted\", and \"ALE\" for consistency with literature. Additionally,\nrelevant references on ALE, such as Peidong Yang’s work on methanol adaptation of S.\novata, should be cited.\nResponse: We appreciate the reviewer's insightful suggestion regarding the\nexperimental approach. In response, we have thoroughly revised the manuscript to\nadopt the established microbial engineering terminology of “adaptive laboratory\nevolution (ALE)” throughout. Specifically, all references to strain domestication have\nbeen updated to “ALE-driven adaptation” or “ALE (adaptive laboratory evolution)”\nwhere contextually appropriate, with consistent citation of the foundational work by\nYang et al. (Nano. Lett. 2022, 22, 5503-5509) on methanol adaptation in S. ovata. This\nterminology alignment better reflects current practices in metabolic engineering while\nmaintaining scientific precision.\n• Gold Nanoparticles (AuNP) & Microbial Exposure:\no How long were the cells exposed to the presence of HAuCl during sequential\n4\nexposure to 1, 1.5, and 2 µM HAuCl solutions?\nevolution (ALE) protocol, A. succinogenes cells were sequentially exposed to HAuCl\n4\nconcentrations of 1.0, 1.5, and 2.0 µM, with each exposure step lasting 12 hours. This\nresulted in a total adaptation period of 36 hours. This gradual exposure strategy was\ndesigned to minimize cytotoxic effects while promoting intracellular accumulation of\nAuNPs and enhancing bacterial tolerance.\no What was the size of the synthesized AuNP? Do the authors believe that plasmonic\neffects could play a role in this Au-containing biohybrid system?\nResponse: Thank you for your insightful question. The average size of the\nintracellularly synthesized Au nanoparticles (AuNPs) was determined to be\napproximately 38 nm, based on statistical analysis of TEM images (Fig. R2-1).\nFig. R2-1 TEM image of thin-sectioned Au@A. succinogenes, the scale bar is 200 nm. The inset\nshows the particle size distribution of the Au NPs.\nTo evaluate the potential contribution of localized surface plasmon resonance\n(LSPR) effects, we conducted UV–vis spectroscopy on both wild-type A. succinogenes\nand Au@A. succinogenes. The maximum absorption peaks were observed at 268 nm\nand 273 nm, respectively (Fig. R2-2). Notably, no characteristic LSPR peak typically\nassociated with AuNPs (commonly near ~520 nm) was detected in the Au-containing\nbacterial samples. This indicates that plasmonic resonance is not prominent in our\nsystem.\nFig. R2-2 UV-vis of A.succinogenes and Au@A.succinogenes.\nThe absence of a detectable plasmonic effect can be attributed to the intracellular\nlocalization of Au NPs, which are embedded within the bacterial cytoplasm and closely\nassociated with the inner membrane. In this biological environment, the dense cellular\nmatrix and surrounding biomolecules likely hinder direct optical access to the\nnanoparticle surface, thereby suppressing LSPR excitation. This shielding effect\nprevents efficient interaction between incident light and the Au NPs, which is necessary\nfor plasmonic activation.\nAs a result, the functional role of Au NPs in our system is not related to light\nharvesting or energy conversion via plasmonic, but rather focused on facilitating\nintracellular electron transport. The Au NPs serve as conductive bridges that enhance\ncharge separation and transmembrane electron transfer, enabling efficient coupling\nbetween the photoelectrode and microbial redox metabolism.\no The authors claim that Au NP accumulate near the cell membrane, but Figure 2e\nappears to show Au NP accumulation mainly in the cytoplasm rather than at the\nmembrane. Additionally, this figure is difficult to interpret due to its size; scaling it\nup could improve clarity.\nResponse: Thank you for your valuable comment. We acknowledge the limitations in\nthe resolution and scale of Fig. 2e and have revised the figure layout in the updated\nmanuscript to improve clarity and interpretability. Regarding the localization of Au\nnanoparticles (AuNPs), although Fig. 2e shows the overall distribution of Au within\nthe bacterial cells, higher-resolution HAADF-STEM and EDS mapping indicate that\nAuNPs are predominantly concentrated near the inner membrane region, rather than\nbeing uniformly dispersed throughout the cytoplasm. This spatial distribution is\nsupported by the mechanism of nanoparticle formation: upon uptake of HAuCl ,\n4\nmembrane-associated reductases—such as cytochrome bd oxidase and NADH-\ndependent reductases—act as primary electron donors for the reduction of Au3+ to Au0.\nThese enzymes are enriched in the inner membrane, where they participate in the\nrespiratory chain and maintain a strongly reductive microenvironment. As a result, Au0\nnucleation preferentially occurs adjacent to the inner membrane, leading to localized\naccumulation of nanoparticles in that region.\nMoreover, this membrane-proximal positioning is consistent with our design\nrationale: the inner membrane-localized AuNPs function as conductive intermediates,\nbridging external electron sources from the photoelectrode with intracellular redox\ncenters. This strategic localization enhances charge transfer efficiency without\ninterfering with core metabolic processes. We hope this expanded explanation clarifies\nthe observed distribution pattern of Au NPs and the rationale behind their membrane-\nassociated accumulation.\n• Hydrogel Layer & Bacterial Attachment:\no What is the porosity of the hydrogel layer? Can bacteria go inside any pores?\nResponse: We sincerely appreciate the reviewer’s insightful question. It is important to\nclarify that the hydrogel layer in our design fundamentally differs from conventional\nbulk hydrogel materials. As clearly demonstrated by the TEM image of the\nNiO@PAA@NHS electrode in Fig. R2-3, the PAA@NHS hydrogel is an ultrathin (7.5\nnm) conformal layer without microporosity, preventing bacterial encapsulation but\nensuring tight interfacial adhesion.\nFig. R2-3 TEM image of NiO@PAA@NHS nanosheets.\nAdditionally, FTIR spectra (Fig. R2-4) confirm covalent bond formation via NHS-\nester reactions with bacterial amine groups. Regarding the claim of covalent linkage,\nwe have added clearer experimental evidence and revised the graphic in Fig. 1 to\nreflect covalent bonding more accurately as a likely, but not yet unambiguously proven,\nmechanism.\nFig. R2-4 Fourier transform infrared spectroscopy (FTIR) of NiO@PAA@NHS/A.succinogenes and\nA. succinogenes.\no How do the authors confirm that NHS has reacted with the amine groups on the\nbacterial surface? This would help support their proposed hypothesis and mechanism,\nlater on.\nResponse: We sincerely appreciate the reviewer's insightful comments. Fig. R2-4 is a\ndetailed analysis of the FTIR spectra. For A. succinogenes, the peaks at 1627 cm-\n1 and 1533 cm-1 were assigned to C=O stretching and N-H bending vibrations,\nrespectively. Similarly, a peak observed at 1043 cm-1 corresponds to C-N stretching\nvibration. In contrast, for NiO@PAA@NHS/A. succinogenes, the peaks for C=O\nstretching and N-H bending vibrations shifted to 1685 cm-1 and 1625 cm-1, respectively,\nshowing a significant blue shift. This indicates increased bond strength, suggesting that\nthe electrode and bacterial cells reacted to form stronger amide bonds.\no The authors claim that A. succinogenes contains dangling NH groups in its\n2\nmembrane-bound cytochromes (Cyt d I and d II), allowing covalent attachment to the\nhydrogel. However, Ref 40 does not provide any information on amines in membrane-\nbound cytochromes. Could the authors provide supporting data to their claim and cite\nappropriate literature references?\nResponse: Thank you for this insightful comment. We acknowledge that Ref. 40 does\nnot directly discuss amine functionalities in cytochrome bd oxidases. To further support\nour claim, we have re-evaluated the structural features of these enzymes based on\nrecent high-resolution studies. Specifically, the crystal structure of cytochrome bd\noxidase from Geobacillus thermodenitrificans, published by Safarian et al. in Science\n(2016), provides crucial structural insight. Their work reveals that cytochrome bd\noxidases, including homologs of Cyt bd-I and Cyt bd-II, are composed of multiple\nmembrane-integrated α-helical domains and exposed surface regions, notably the Q\nloop, which harbors lysine (Lys252, for example) and glutamate residues that are\nsolvent-accessible. Lysine residues contain primary amine (-NH ) groups, which are\n2\nchemically reactive with NHS esters and thus are plausible sites for covalent\nconjugation to our NHS-functionalized hydrogel layer. Given the conserved topology\nand function of cytochrome bd complexes across species, it is highly likely that A.\nsuccinogenes possesses similar membrane-bound cytochromes with surface-exposed\namine-containing residues, especially within or near their extracellular domains.\nMoreover, FTIR analysis in our work shows distinct spectral shifts corresponding\nto amide bond formation (Fig. R2-4), further supporting covalent coupling between\nbacterial surface proteins and the hydrogel.\nReference: Safarian, S. et al. Structure of a bd oxidase indicates similar mechanisms for membrane-\nintegrated oxygen reductases. Science 352, 583–586 (2016).\nhttps://doi.org/10.1126/science.aaf2477\no The manuscript frequently refers to \"bacterial adhesion\" and \"surface\nfunctionalization\" as key factors in improving attachment and not covalent linkage.\nHowever, Figure 1 depicts the bacteria as covalently linked to the hydrogel, yet no\nexperimental evidence confirms this covalent bonding (bacteria-NH-CO-hydrogel).\nUnless further characterization proves covalent attachment, I strongly recommend the\nauthors tone down their claims and modify Figure 1 (and any related figures)\naccordingly.\nResponse: We thank the reviewer for this important and constructive comment. We\nagree that rigorous distinction between non-covalent adhesion and covalent\nimmobilization is essential, both in the text and in figure representations. In our study,\nthe use of NHS-functionalized hydrogel coatings was specifically intended to facilitate\ncovalent bonding with primary amine groups on the bacterial surface. Supporting\nevidence comes from FTIR spectroscopy (Fig. R2-4), which shows clear shifts in the\namide I and II regions, consistent with the formation of amide bonds (–NH–CO–)\nbetween NHS esters and –NH₂ groups on membrane-associated proteins. In the\nmanuscript text, we have updated our wording to clarify that covalent bonding is\nsuggested by spectroscopic evidence, but not yet directly proven at the molecular level.\nWe appreciate the reviewer’s suggestion, which has helped us improve the clarity and\nscientific rigor of both our claims and visual representations.\no Cell Density in Figure 2j: I could not find details in the methods section regarding\nhow the authors quantified the number of cells per mm². This information should be\nincluded.\nResponse: Thank you for this helpful suggestion. To quantify the bacterial cell density\nshown in Figure 2j, we employed the following standardized procedure: High-\nresolution SEM images were acquired from five randomly selected fields of view on\neach electrode surface. The images were analyzed using ImageJ software, where\nbacterial cells were identified through threshold segmentation, followed by manual\ncorrection to ensure accuracy. The actual surface area of each field was calculated\nbased on the scale bar, and bacterial counts were normalized accordingly to yield cells\nper mm2. The reported data represent the mean ± standard deviation of five independent\nregions. We have now added a detailed description of this quantification method in the\nMethods section of the revised manuscript to enhance transparency and reproducibility.\nWe sincerely appreciate the reviewer’s recommendation, which helped us clarify this\nkey methodological aspect.\n• Photoelectrochemical (PEC) Experiments:\no What are the faradaic efficiency of all products (coming from the (photo)electrodes\nand bacteria) and CO conversion efficiency to succinate?\nor photoelectrocatalytic systems, the PEC-biohybrid platform described in our study\nintegrates both electrochemical electron injection and biological carbon metabolism.\nIn this system, succinic acid is produced not solely via CO electroreduction, but\n2\nthrough a complex bioconversion process involving both glucose metabolism and CO₂\nfixation within the microbial TCA cycle.\nDue to the presence of glucose as a major carbon substrate and the synergistic\nenhancement of microbial activity under PEC conditions, the overall succinate\nproduction was significantly increased. When we attempted to calculate Faradaic\nefficiency based solely on succinate output, the resulting values exceeded 100%,\nwhich is a clear indication that traditional Faradaic efficiency calculations—typically\napplied to purely abiotic electrochemical systems—are not directly applicable to this hybrid\nbio-electrochemical context.\nTherefore, to better evaluate carbon utilization, we instead calculated the CO₂\nconversion efficiency, which reflects the proportion of CO₂ fixed into succinate. The\nNiO@PAA@NHS/Au@A. succinogenes system achieved a CO -to-succinate\n2\nconversion efficiency of 67% (Fig. R2-5).\nFig. R2-5 CO conversion rate of NiO@PAA@NHS/Au@A. succinogene, NiO@PAA@NHS A.\n2\nsuccinogene, NiO/A. succinogene and A. succinogene.\no Were there any byproducts, such as H or CO, formed at the photocathodes?\nthat under the applied potential of –0.3 V vs. RHE, the NiO@PAA@NHS/A.\nsuccinogenes photocathode system does indeed generate minor amounts of H and CO\n2\nas byproducts. Hydrogen (H ) is produced via partial water reduction at the\n2\nphotocathode surface. As shown in Fig. R2-6a, the amount of H generated after 8\n2\nhours under AM 1.5G illumination is relatively low, reaching only 144 μmol.\nCarbon monoxide (CO) is produced as a transient intermediate during CO\n2\nreduction. In the normal PEC-biohybrid operating conditions, CO is rapidly consumed\nwithin the microbial metabolism and does not accumulate significantly.\nWe would like to emphasize that during gas-phase product analysis, a sealed system\nwas used for gas chromatography (GC) measurements to enable accurate\nquantification. Due to the closed nature of this setup, continuous CO bubbling was not\n2\npossible. Instead, a large volume of CO was introduced at once prior to the reaction,\n2\nleading to a high initial CO concentration in the headspace. This atypical condition\n2\ndiffers from the standard PEC-biohybrid operating setup, where continuous CO supply\n2\nand open exchange with the environment allow steady-state concentrations. Under the\nsealed GC conditions, the elevated CO partial pressure promotes a higher initial rate\n2\nof CO formation. As a result, the CO yield reached 425.7 μmol after only 3 hours of\nPEC operation (Fig. R2-6b), which is significantly higher than what occurs under\nnormal reaction conditions. Thus, while CO production is observed under GC testing,\nit does not accurately represent the behavior of the PEC-biohybrid system during\nstandard operation, where CO accumulation remains minimal due to ongoing microbial\nuptake and downstream conversion.\nFig. R2-6 (a) H and (b) CO production on NiO@PAA@NHS/Au@A. succinogenes\n2\nduring PEC reaction at -0.3 V (vs RHE).\no The authors show that microbes can produce succinate without CO . If PBS medium\n2\nwas used as the electrolyte, was glucose also present as a carbon source in the PEC\nexperiments?\nResponse: Thank you for raising this important point. We would like to clarify that our\nstudy does not imply that succinate can be produced independently of CO . In fact, CO\n2 2\nis a critical co-substrate in the metabolic pathway leading to succinate formation in A.\nsuccinogenes, particularly via the reductive branch of the TCA cycle.\nIn earlier experiments conducted under an argon atmosphere, succinate\nproduction was still observed at a rate of 0.566 g/L/h. This phenomenon can be\nattributed to the presence of Mg (OH) CO (15 g/L) in the fermentation medium, which\n2 2 3\nserved dual purposes: pH buffering and providing a bioavailable source of CO through\n2\ncarbonate equilibrium. Therefore, even under nominally CO -free conditions, residual\n2\ninorganic carbon was still available to support succinate synthesis.\nFig. R2-7 Succinic acid yield rate on NiO@PAA@NHS/Au@A. succinogenes biohybrid in CO -\n2\ns Ar-saturated solutions with and without Mg (OH) CO in PEC model at -0.3 V vs RHE.\n2 2 3\nTo further validate this, we performed additional experiments using a modified\nmedium with/without Mg (OH) CO . As shown in Fig. R2-7, under argon and in the\n2 2 3\nabsence of external CO sources, only a small amount of succinate was produced, which\n2\nwe attribute to endogenous carbon reserves within the bacterial cells accumulated\nduring prior cultivation. Notably, succinate levels plateaued after ~4 hours, indicating\nthat sustained production requires exogenous CO input. Regarding the use of PBS as\n2\nthe electrolyte in the PEC experiments: although it lacks additional nutrients, we\nconsistently supplemented all PEC reaction media with 30 g/L glucose, which served\nas the primary organic carbon source and ensured sufficient energy supply and biomass\nsupport for microbial activity.\nWe have clarified these experimental conditions and added the relevant\nexplanations in the revised manuscript to eliminate potential ambiguity regarding the\ncarbon source and the role of CO₂ in succinate biosynthesis.\no The manuscript should explicitly state the carbon source used when Ar is present, and\nthe methods section should clarify the media and conditions used for PEC experiments.\nResponse: Thank you for your thoughtful comment. We would like to clarify that in all\nexperiments conducted under argon (Ar) atmosphere, glucose was consistently used as\nthe sole organic carbon source. This was maintained across both control and PEC\nconditions to ensure comparability. To assess the contribution of inorganic carbon, we\nperformed parallel experiments with and without the addition of Mg (OH) CO , which\n2 2 3\nacts as both a pH buffer and a reservoir of CO through dissolution and equilibrium.\nthe role of CO availability in sustaining succinate production. Furthermore, we\n2\nconfirm that the electrolyte composition in all PEC experiments was identical to the\nfermentation medium described in the Methods section, unless otherwise noted. We\nhave now explicitly stated this information in the revised manuscript to improve clarity\nand reproducibility.\no The manuscript lacks experimental data and explanations for electrochemical\nbiohybrids (EC) and photochemical biohybrids (PC) (Figure 3b).\nResponse: Thank you for your thoughtful comment regarding Fig. 3b. We appreciate\nthe opportunity to clarify the experimental conditions: The electrochemical biohybrid\n(EC) system was operated at -0.3 V vs. RHE under dark conditions. The photochemical\nbiohybrid (PC) system was evaluated under AM 1.5G illumination (100 mW/cm2). We\nselected these conditions to enable direct comparison between electrical and optical\nstimulation approaches. We sincerely appreciate your suggestion and will include these\nimportant experimental details in the revised manuscript.\no Regarding Figure 3b, the text only mentions the succinate formation rate, but without\nadditional data, it is unclear why the EC-biohybrid system performs worse than the\nPEC-biohybrid system.\nResponse: We thank the reviewer for this insightful question regarding the performance\ncomparison in Fig. 3b. While both the photoelectrochemical (PEC) and\nelectrochemical (EC) biohybrid systems were operated at an applied potential of –0.3\nV vs. RHE, the superior performance of the PEC system can be attributed to the\nsynergistic effect of light irradiation and electrochemical bias.\nSpecifically, the PEC system, under AM 1.5G illumination, generates a\nsignificantly higher and more stable current than the EC system operating in the dark.\nThis enhancement reflects more efficient photo-induced charge carrier generation,\nwhich leads to greater electron availability for microbial uptake, thereby accelerating\nsuccinate biosynthesis. The improved electron flux in the PEC system promotes a higher\nintracellular NADH/NAD+ recycling rate, supports more active redox metabolism, and\nupregulates key genes in the CO assimilation and succinate synthesis pathways, as\n2\nshown in Figs. 4d–f. We appreciate the reviewer’s comment, which has allowed us to\nclarify this important distinction.\no Biohybrid experiments typically run for dozens of hours or days, yet the PEC\nchronoamperometry experiments presented here last only 2 hours. How long were the\nbiohybrid experiments run for Figures 3b, 3c, and 3e?\nResponse: We sincerely thank the reviewer for this important observation regarding\nexperimental duration. We would like to clarify that the chronoamperometry experiment\nshown in Fig. 3d was specifically designed as a short-term test (2 hours) to rapidly\nassess photocurrent response profiles under varying applied potentials.\nIn contrast, the biohybrid performance experiments presented in Figs. 3b, 3c, and\n3e were each conducted over a 24-hour period, which aligns with standard timeframes\nin biohybrid photoelectrochemical systems. These longer durations were chosen to\nenable a more comprehensive evaluation of succinate accumulation, system stability,\nand catalytic consistency under sustained reaction conditions.\nTo improve clarity, we have now explicitly stated the exact duration of each\nexperiment in the figure captions and Methods section of the revised manuscript. We\nare grateful for the reviewer’s suggestion, which has helped us enhance the\ntransparency and reproducibility of our work.\no In Supplementary Figure 14, how long is each cycle? If it is 10 hours per cycle, this\nshould be explicitly stated for clarity.\nResponse: Thank you for the reviewer’s comment. In Supplementary Fig. 14, the initial\nexperiments were conducted with 10-hour cycles. To further assess the long-term\noperational stability of the PEC-biohybrid system, we extended the experiments to\ninclude continuous light/dark cycling over a period of 5 days (Fig. R2-8).\nFig. R2-8 Succinic acid yield rate on NiO@PAA@NHS/Au@A. succinogenes biohybrid for 5 days\nof reactions. Data are represented as means ± SD (n=3).\nThese extended tests more clearly demonstrate the system’s robustness during\nprolonged operation. Nevertheless, we observed a gradual decline in succinate\nproduction after 5 days, which we attribute to minor degradation of the electrode\nmaterials and a reduction in bacterial viability under sustained PEC stress conditions.\nWe have now explicitly stated the cycle duration and clarified these experimental\ndetails in the revised manuscript to enhance clarity and reproducibility.\no Figure 3g and Supplementary Figure 14 lack error bars—were these plots generated\nfrom triplicate experiments?\nResponse: We sincerely thank the reviewer for this valuable suggestion. In the revised\nmanuscript, we have updated Fig. 3g (Fig. R2-9) and Supplementary Fig. 14\n(Supplementary Fig. 19 in revision) to explicitly indicate that the long-term stability\ntests were conducted with three independent biological replicates.\nFig. R2-9 Long-term (5 days) succinic acid generation on NiO@PAA@NHS/Au@A.\nsuccinogenes biohybrid in alternating PEC and EC models at -0.3 V vs RHE. Data are\nrepresented as means ± SD (n=3).\nError bars representing the standard deviation have been added to the plots to\nreflect experimental variability. We appreciate the reviewer’s attention to this detail,\nwhich has helped us further improve the rigor and clarity of our data presentation.\n• Electron Transfer Pathway & Proposed Mechanism:\no Figure 4 clearly depicts the metabolic pathway for microbial CO and glucose\n2\nconversion to succinic acid. However, the extracellular electron transfer pathway\nremains unclear. No data (e.g., spectroscopy results) support the hypothesized\nextracellular electron transfer mechanism. One suggestion is to use time-resolved\nabsorption or vibrational spectroscopy to determine whether the AuNP are\nreduced/oxidized by internal microbial electron transport chains.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion. In response,\nwe employed time-resolved fluorescence spectroscopy to investigate the extracellular\nelectron transfer process.\nFig. R2-10 Fluorescence lifetime spectra in different systems.\nThe results (Fig. R2-10) showed that the A. succinogenes@Au biohybrid system\nexhibited a fluorescence lifetime of 1.89 ns, representing a 45% reduction compared to\nthe wild-type strain. This significant lifetime decrease confirms enhanced electron\ntransfer efficiency between intracellular AuNPs and the microbial electron transport\nchains. The corresponding data and detailed analysis have been included in the revised\nmanuscript to further substantiate the proposed extracellular-to-intracellular electron\ntransfer mechanism.\no Based on Ref 40, A. succinogenes contains membrane-bound cytochromes and\nhydrogenases. Given the (photo)currents at -0.3 V vs. RHE, it is likely that the microbes\nare not able to uptake all charges and use instead in situ-produced H from the\n2\n(photo)electrodes as an electron donor for succinic acid production. Therefore, a key\ncontrol experiment to test this point would be using a knockout strain lacking\nmembrane-bound hydrogenases to see whether succinic acid production is affected\nunder the same conditions.\nResponse: We sincerely appreciate the reviewer’s insightful comment. We fully agree\nthat knockout of membrane-bound hydrogenases would be a definitive approach to\nclarify the role of H uptake. However, given the complexity of genetic manipulation in\nan experiment was not feasible within the scope and timeline of the current study.\nNevertheless, we addressed this point through an alternative, practical approach: by\ncomparing H production between the NiO@PAA@NHS electrode with and without\n2\nbacterial inoculation under identical PEC conditions.\nFig. R2-11 H production in NiO@PAA@NHS and NiO@PAA@NHS/A. succinogenes@Au.\nH when A. succinogenes was present, indicating that the bacteria actively consumed\n2\nthe in situ-produced H generated at the photocathode surface. Thus, although a\n2\nhydrogenase-deficient mutant would provide further mechanistic insights, our\nexperimental evidence already supports the conclusion that A. succinogenes can\neffectively utilize photogenerated H as an auxiliary electron donor for enhanced\n2\nsuccinic acid production.\no To confirm that succinic acid is derived from CO (and not solely from glucose),\n2\n13CO and 13C-glucose labelling experiments should be conducted.\nsource contributions to succinic acid production, we conducted dual isotopic labeling\nexperiments using 13CO and 13C -glucose (13C H O ), followed by 1H-NMR and 13C-\n2 6 6 12 6\nNMR analyses.\nFig. R2-12 (a) 1 H-NMR and (b) 13C-NMR spectra of succinic acid labeled with 13C.\nIn the 1H-NMR spectra (Fig. R2-12a), the methylene proton signal of succinic acid\nappears at 2.34 ppm. Upon 13CO labeling, this peak remained unchanged, suggesting\n2\nthat 13CO incorporation does not affect proton splitting at this site. However, when\n2\nusing 13C -glucose as the substrate, the methylene proton peak split into a doublet, due\n6\nto 13C–1H coupling, indicating successful incorporation of 13C from glucose into the\nsuccinate carbon skeleton through glycolytic and TCA metabolic pathways.\nIn the 13C-NMR spectra (Fig. R2-12b), the carboxyl carbon (-COOH) resonated\nat approximately 181 ppm, and its signal intensity increased significantly after 13CO\n2\nlabeling, providing direct evidence that external CO contributes to the formation of\n2\nthe carboxyl groups in succinic acid. Additionally, the methylene carbon (-CH -) signal\n2\nobserved at 32 ppm showed enhancement after both 13CO and 13C -glucose labeling,\n2 6\nconfirming that both CO fixation and glucose metabolism contribute to the assembly\n2\nof the succinate backbone.\nTogether, these dual-labeling NMR results clearly demonstrate that succinate\ncarbon originates from both CO and glucose, with glucose primarily supplying the\n2\ncarbon skeleton (through glycolysis and partial TCA metabolism) and CO directly\n2\nfixing into the carboxyl groups during reductive biosynthesis. We have included these\ndetailed findings in the revised manuscript to strengthen the mechanistic understanding\nof carbon flow in our PEC-biohybrid system.\n• References:\no References 30 and 31 are incorrect—please verify them and check the rest.\nResponse: We sincerely thank the reviewer for their meticulous attention to detail. We\nhave corrected the reference citation, and carefully checked full reference list. We\nappreciate the reviewer’s diligence, which has helped us further strengthen the quality\nand reliability of our submission.\nReviewer #3 (Remarks to the Author):\nThe authors demonstrate a certain range and novelty compared to the current state\nof the art. However, I must state that the manuscript, in its current form, does not meet\nthe journal's standards. Please see the comments below for the reasons behind this.\nNevertheless, I see potential for the manuscript to meet these standards, provided the\ncomments are addressed.\nResponse: We sincerely thank the reviewer for their positive and encouraging\nevaluation of our work. We highly appreciate your recognition of the potential value of\nour manuscript. Following your insightful and detailed comments, we have carefully\nrevised the manuscript and provided point-by-point responses addressing each of your\nsuggestions. Your feedback has been instrumental in helping us significantly improve\nthe scientific rigor, clarity, and overall quality of the paper. Once again, we sincerely\nthank you for your constructive review and valuable guidance, which have been critical\nto enhancing this work.\nMajor points:\nThe text describes the evaluation of linear sweep voltammetry between Ar- or CO₂-\nsaturated electrolyte under the influence of light or in darkness. Consequently, a\n“significantly enhanced” current density is reported both in the light and in the dark\nunder a CO atmosphere. However, two points need to be critically examined: Firstly,\n2\nthe difference between the two experiments conducted in darkness is relatively small.\nSecondly, the results presented appear to be based on a single measurement (n = 1),\nwhich does not allow for any statistically reliable conclusion to be drawn about the\nobserved differences. This should be clearly stated in the discussion. Additionally, the\nstatement on succinate yields under CO or Ar conditions is redundant. It is well known\n2\nthat succinate production in A. succinogenes is CO -dependent via the reductive\n2\nbranches of the TCA cycle (see work by James B. McKinlay).\nResponse: Thank you for your professional and thoughtful comments. Regarding the\nLSV experiments conducted under CO - and Ar-saturated conditions, we would like to\n2\nclarify that multiple scans were performed for each condition to ensure reproducibility.\nThe final curves presented in the manuscript represent consistent trends observed\nacross repeated measurements, although statistical error bars were not included in the\ninitial submission. We have now revised the discussion to explicitly acknowledge this\nlimitation and to emphasize that conclusions were drawn based on reproducible\nexperimental trends rather than a single measurement.\nAs for the comparison of succinate production under CO and Ar atmospheres, we\n2\nfully agree with the reviewer that CO -dependent succinate biosynthesis via the\n2\nreductive branch of the TCA cycle is well established, as shown in the works of James\nB. McKinlay (Metab. Eng. 9, 177-192 (2007), Metab. Eng. 10, 55-68 (2008), BMC\nGenomics 11, 680 (2010)). In our study, this comparison was not intended to claim\nnovelty regarding the biological mechanism, but rather to confirm that the PEC-\nbiohybrid system indeed drives CO₂ fixation into succinate under the specific\nengineered conditions, thus supporting the overall functional operation of the system.\nWe have cited the corresponding reference papers from James B. McKinlay, and revised\nthe corresponding discussion in the manuscript to reflect these points and avoid\nredundancy more clearly.\nIn general, the manuscript and the figures lack precise information on the statistics,\nparticularly regarding the sample size. For instance, some diagrams in Fig. 3/4 display\na standard deviation, while others do not. There is no explanation for this either in the\ncaptions or in the text. It also remains unclear whether the data are technical or\nbiological replicates. Without this information, it is difficult to assess the reliability of\nthe results, which significantly hinders interpretation, especially as the text frequently\nrefers to “significant” changes. This needs to be clarified.\nResponse: Thank you for the reviewer’s critical and important comment regarding the\nstatistical treatment of the data. We would like to clarify that all quantitative\nexperiments were conducted using biological triplicates (n = 3). In the revised\nmanuscript, we have now explicitly indicated the sample size in the figure captions and\ndistinguished between biological replicates and technical replicates wherever\nappropriate. Additionally, we have added standard deviation bars to all relevant figures\nto ensure consistent and transparent presentation of data variability. We have also\nupdated the manuscript text to use terms like \"significant changes\" more cautiously,\nensuring that they are supported by appropriate statistical context. We sincerely\nappreciate the reviewer’s comment, which has helped us improve the clarity, reliability,\nand scientific rigor of our data presentation.\nRegarding the results in Fig. 3g, the data shown correspond to a light-dark cycle\nintended to simulate a day-night rhythm. The simulations indicate low productivity\nduring the night phases. This implies that the improvement in succinate production rates\n(g/L/h) emphasised in this manuscript actually occurs only during the day phase. Over\na multi-day fermentation process, the average productivity would therefore be lower,\ncalling into question the claimed “superior efficiency” of the system. This limitation\nshould be acknowledged in the discussion.\nResponse: Thank you for the reviewer’s insightful comment regarding the diurnal\nvariation in productivity illustrated in Fig. 3g. We fully acknowledge that succinate\nproduction rates decline significantly during the dark (electrochemical) phase\ncompared to the light (photoelectrochemical) phase. Consequently, over multi-day\nfermentation periods, the average productivity would indeed be lower than the peak\nrates achieved during illumination.\nHowever, as shown in Fig. 3b, even when accounting for the reduced productivity\nduring the night phase, the overall system performance under PEC/EC operation still\ndemonstrates a threefold improvement in succinate production rate compared to\nconventional microbial fermentation methods. This reflects a substantial advance\nrelative to existing biological systems.\nIn response to the reviewer’s comment, we have revised the manuscript discussion\nto clearly state that the \"superior efficiency\" claim refers specifically to the peak\ndaytime (light phase) productivity, and we now openly acknowledge the system's light-\ndependent limitations over extended operations. We sincerely appreciate the reviewer’s\nsuggestion, which has helped us present a more balanced and transparent evaluation\nof our system’s advantages and areas for future improvement.\nIn general, it would be of interest to analyse the results over a complete fermentation\nrun rather than solely the initial hours. In addition to concentration data, the yield in\nterms of metabolised glucose (g/g) should also be considered. This would indicate\nwhether not only the production rate of succinate (g/L/h) but also the overall yield (g/g)\nis improved. In other words, it would determine whether more carbon is actually\nconverted into the target product, succinic acid. This additional consideration would be\ncrucial in fully evaluating the efficiency of the process.\nResponse: Thank you for your expert feedback. In response to your suggestion, we have\nevaluated the glucose-to-succinate yield (g/g) over a complete fermentation cycle (Fig.\nR3-1).\nFig. R3-1 Glucose conversion rate of NiO@PAA@NHS/Au@A. succinogene, NiO@PAA@NHS A.\nsuccinogene, NiO/A. succinogene, and A. succinogene.\nThe NiO@PAA@NHS/Au@A. succinogenes system achieved a glucose conversion\nyield of 0.78 g/g, representing an approximately 20% improvement compared to the\nwild-type A. succinogenes control. This additional analysis confirms that, beyond\nenhancing the succinate production rate (g/L/h), our system also significantly improves\noverall carbon utilization efficiency toward the target product. We have incorporated\nthese results into the revised manuscript to provide a more comprehensive assessment\nof process efficiency. We sincerely appreciate the reviewer’s suggestion, which has\nstrengthened the evaluation of our system’s performance.\nThe manuscript lacks a critical discussion of the challenges posed by the use of light in\nlarge-scale applications, despite the method being lauded in the abstract. Typical stirred\ntank reactors for industrial applications would either require integrated light sources or\nbe replaced by alternative, scalable reactor systems capable of supporting both the\nelectrochemical (electrodes) and photochemical components. At present, the scale-up\nof bioelectrochemical systems remains a significant challenge. Moreover, research on\nphototrophic organisms, such as cyanobacteria, has demonstrated that incorporating\nlight into biotechnological processes presents considerable obstacles, including limited\nlight penetration in the reactor (restricting reactor diameter), shading issues, and more.\nThese challenges should be addressed in the discussion and conclusion, especially since\nthe system is described as a “leading candidate for large-scale production and\ncommercialisation.”\nResponse: We sincerely thank the reviewer for this valuable and important comment.\nWe fully acknowledge that scaling up light-driven biohybrid systems for industrial\napplications presents substantial challenges, particularly regarding light distribution,\nreactor geometry, and energy efficiency. These are critical issues that must be addressed\nbefore commercialization can be realized.\nRecent advances, however, provide promising strategies to overcome these\nlimitations. For example, computational fluid dynamics (CFD)-optimized plate-type\nairlift photobioreactors have demonstrated enhanced light utilization efficiency and\nreduced energy consumption, offering viable design options for scalable\nphotobiocatalysis (Engineering 3, 318–329 (2017)). Furthermore, successful scale-up\nexamples in cyanobacterial systems have shown that closed photobioreactors (PBRs)\ncombined with continuous cultivation strategies can maintain high productivity with\nsustainable energy inputs (Renew. Sustain. Energy Rev. 154, 111852 (2022)).\nKey engineering considerations include achieving uniform illumination, optimized\nmixing patterns, and high biomass retention. For instance, transparent grid-column\nPBRs have been used to enable continuous hydrogen production with improved light\npenetration, while Box-Behnken Design (BBD) optimized operational parameters have\nbeen applied to maximize light absorption and biochemical productivity (Bioresour.\nTechnol. 291, 121806 (2019)). Additionally, Palamae et al. demonstrated that tubular\nphotobioreactors, which offer a high surface-area-to-volume ratio, outperform other\ngeometries for bacterial growth and hydrogen production (J. Clean. Prod. 181, 318–\n328 (2018)).\nAlthough significant challenges remain, these technological advances — including\nreal-time parameter control, modular reactor design, and optimized light distribution\nsystems — suggest a progressive pathway toward industrialization of photo-biohybrid\nsystems. We have incorporated this critical discussion into the revised manuscript to\nprovide a more balanced and realistic perspective on the opportunities and challenges\nassociated with large-scale application. We sincerely appreciate the reviewer’s\ncomment, which has helped us significantly improve the depth and completeness of our\ndiscussion.\nThe section from “Upon the absorption of the substrate glucose by the bacteria, it is\nconverted into phosphoenolpyruvate (PEP), marking the beginning of the metabolic\ndivergence into two distinct pathways.” to the end of the paragraph serves as a general\nsummary of the metabolism of A. succinogenes. This content is more appropriate for\nthe introduction rather than the results or discussion sections. Furthermore, this section\nlacks references to substantiate the metabolic processes described. Appropriate sources\nmust be included.\nResponse: Thank you for your valuable suggestion. We agree that the section\nsummarizing the general metabolic pathways of A. succinogenes would be more\nappropriately placed in the Introduction rather than within the Results or Discussion\nsections. Accordingly, we have relocated this content to the Introduction in the revised\nmanuscript. Additionally, to strengthen the mechanistic analysis and support the\ndescribed metabolic processes, we have incorporated two appropriate references as\nsupporting literature (Fermentation, 9, 1026 (2023), Appl. Microbio. Biotechnol. 100,\n9641-9652 (2016)). We sincerely appreciate the reviewer’s insightful comment, which\nhas helped us improve both the structure and the scientific rigor of the manuscript.\nGene expression analysis of key metabolic enzymes shows an upregulation of all\nstudied enzymes compared to the control, including the enzymes PK, ACK, and PfIB,\nwhich are involved in by-product reactions. Consequently, it would be important to\ncompare the yields of by-products between the control and the biohybrids. The text\nmerely states: “Although the concentrations of acetic and formic acids increased,” yet\nconcrete data are missing. By what proportion did the by-products increase? Was there\na shift in the ratio between by-products and main products? These aspects are not\ndiscussed further, despite the upregulation of the relevant enzymes. This section should\nbe expanded upon, and the corresponding data on by-products should be included.\nResponse: We sincerely appreciate the reviewer’s insightful comments regarding the\nneed for a more detailed analysis of product distribution in our study. In response,\nwe have conducted a comprehensive comparison of the yields and proportions of the main\nproduct (succinic acid) and byproducts (acetic acid, formic acid, and pyruvic acid)\nbetween the control and PEC-biohybrid systems (Fig. R3-2). Due to metabolic\ninteractions within the microbial network, the enhanced succinate production was\naccompanied by synchronous increases in the production of these byproducts.\nFig. R3-2 (a) Yield rate of succinic acid, acetic acid, formic acid, and pyruvic acid on\nNiO@PAA@NHS/Au@A. succinogenes biohybrid. Data are represented as mean ± SD (n=3).\n0.01<*p<0.05, 0.001<**p<0.01, ***p<0.001;(b) Product distribution proportion diagram after\n24 hours of PEC (NiO@PAA@NHS/A. succinogenes and NiO@PAA@NHS/Au@A. succinogenes)\nreaction and PC reaction (A. succinogenes).\nQuantitative analysis revealed that after applying the PEC catalytic process: The\nproportion of succinic acid relative to total organic acids increased from 44% (control)\nto 52% (PEC-biohybrid). The absolute concentrations of acetic acid, formic acid, and\npyruvic acid also increased; however, the ratio of succinic acid to total byproducts\nimproved, indicating a shift favoring the desired product.\nAlthough the incorporation of Au NPs significantly enhanced the overall succinate\nyield, the relative distribution pattern among the metabolic products showed only\nmoderate changes, suggesting that the PEC stimulation primarily boosts overall\nmetabolic flux rather than completely redirecting carbon flux toward a single product.\nWe have now included these detailed results and the corresponding discussion in\nthe revised manuscript to provide a clearer and more complete picture of the system’s\nmetabolic outcomes. We thank the reviewer again for this important suggestion, which\nhas helped us strengthen the rigor and completeness of our metabolic analysis.\nMinor points:\nThe manuscript lacks headings and subheadings to provide a clearer structure.\nThe division of individual figures into up to ten sub-figures, each employing very\ndifferent methods (e.g., diagrams, microscopic images from different microscopes),\nmakes the manuscript difficult to follow and diminishes readability. To enhance clarity,\nthese illustrations should be divided into multiple smaller figures and presented\nsequentially within the text.\nResponse: We sincerely thank the reviewer for their constructive comments regarding\nthe manuscript’s structure and figure organization. We have restructured the\nmanuscript by introducing clearer headings and subheadings throughout the Results\nand Discussion sections to improve logical flow and readability. We would like to clarify\nthat each major figure in the manuscript was carefully designed to center around a\nsingle coherent scientific theme, with the associated sub-figures presenting\ncomplementary datasets obtained through different methodologies (e.g., microscopy,\nelectrochemical analysis, or spectroscopic characterization). This integrated approach\nwas intended to provide a comprehensive view of each key experimental topic in a\nlogically connected manner. We appreciate the reviewer’s feedback, which has helped\nus improve both the structural organization and the accessibility of the manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nNo more questions.\nResponse: Thank you very much for your thorough review and for stating that you have\nno further questions. We appreciate your time and constructive feedback, which has\nhelped us improve the manuscript.\nReviewer #2 (Remarks to the Author):\nI thank the authors for their detailed responses. As a follow-up to the previous round of\ncomments, I would like to raise the following additional points and questions:\nResponse: Thank you very much for your continued engagement and for the additional\nquestions and suggestions. We appreciate your efforts in helping us further improve the\nquality of the manuscript. In response, we have carefully addressed each of the new\npoints raised and have provided detailed replies below.\nCO₂ Conversion Efficiency\nThe method used to calculate the CO₂ conversion efficiency is not described. Does this\ncalculation account for the total amount of CO₂ present in the system at the start of the\nexperiment? Please clarify how this metric was derived.\nResponse: Thank you for highlighting the need for a full description of the CO₂-\nconversion calculation. In the revised manuscript we have added an explicit subsection\nin “Methods” entitled “Determination of CO₂ conversion efficiency” that now reads:\nCO₂ flow rate was monitored continuously with a calibrated mass-flow meter and fixed\nat 7 mL min-1 (at 298 K and 1 atm). The molar CO₂ feed rate was therefore ṅ =\n(CO₂, input)\n7 mL min-1 ÷ 24450 mL mol-1 = 2.86 × 10-4 mol min-1. Succinic acid was detected by\nHPLC; its molar yield was quantified at different reaction durations. The CO₂\nconversion efficiency was then calculated as:\n𝑆𝑢𝑐𝑐𝑖𝑛𝑖𝑐 𝐴𝑐𝑖𝑑 𝑌𝑖𝑒𝑙𝑑(𝑚𝑜𝑙)\nCO Conversion Rate (%) =( )×100%\n2\n𝐶𝑂2 𝐼𝑛𝑝𝑢𝑡 (𝑚𝑜𝑙)\nReporting of Succinate Concentration\nCould the authors report the molarity of succinate produced in addition to the current\nunits (g/L/h)? This would aid in comparison with other systems. Additionally, including\nthe volume of medium used during the experiments would help readers better\nappreciate the scale of the reactor and the biohybrid system.\nResponse: We thank the reviewer for this helpful suggestion. In the revised manuscript\nwe now report succinate concentration in both g L-1 h-1 cm-2 and mol L-1 h-1 cm-2: the\nmeasured productivity of 1.41 ± 0.04 g L-1 h-1 cm-2 corresponds to 0.012 ± 0.0003 mol\nmol L-1 h-1 cm-2 (M = 118.09 g mol-1). This additional value has been added in Revision.\nWe have also added the working volume (50 mL in a 100 mL reactor) to the\n“Experimental setup” section to facilitate scale comparisons with other biohybrid\nsystems.\nFaradaic Efficiency (FE)\nWhile I understand the challenges in determining Faradaic efficiency, it remains a\ncritical metric in evaluating (photo)electrochemical systems, including biohybrids.\nCould the authors report the FE for CO₂-to-succinate conversion and provide a\njustification for any FE values exceeding 100%? The authors could use their response.\nResponse: We sincerely appreciate the reviewer’s emphasis on Faradaic efficiency (FE)\nas a key metric for (photo)electrochemical systems. We acknowledge that FE\nvalues >100% in our system warrant clarification and justification. Below, we address\nthis observation and contextualize its relevance to biohybrid systems:\nThe apparent FE >100% arises from two synergistic factors: (1) Dual Carbon\nSources: Isotope labeling (13CO₂ and 13C -glucose) confirmed that the carbon\n6\nbackbone of succinic acid derives from both CO₂ and organic substrates (glucose) R1.\nFE calculations typically assume all product carbon originates from CO₂ reduction,\nbut in our biohybrid, glucose metabolism contributes significantly to the carbon\nskeleton. Thus, FE normalization solely to CO₂-derived electrons inflates the value\nbeyond 100%. (2) Synergistic Electron Utilization: Electrons from the photoelectrode\nsynergize with microbially generated reducing equivalents (e.g., NADH from glucose\ncatabolism) R2. This dual electron input enhances succinate yields beyond what is\nachievable via electrochemical reduction alone, leading to an apparent FE >100%\nwhen only external circuit electrons are considered.\nWhile FE is a well-established metric for abiotic electrocatalysis, its applicability\nto microbial biohybrids requires careful interpretation: FE quantifies charge efficiency\nonly for the electrochemical reaction. In biohybrids, microbial metabolism\nindependently generates reducing power (e.g., via glycolysis), which supplements\nphotoelectrode-derived electrons. This creates a \"FE inflation\" effect when product\nformation is attributed solely to electrochemical inputs. To our knowledge, no prior\nstudies report FE for microbial electrosynthesis of succinic acid R3,R4. This gap\nhighlights the challenge in defining FE for systems where electrochemical and\nmetabolic pathways are intertwined.\nGiven these complexities, we propose complementary metrics better suited for\nbiohybrids: Our system achieves 67% efficiency, directly quantifying CO₂ fixed into\nsuccinate. Total carbon utilization (glucose + CO₂ → succinate) reached 0.78 g/g ,\nreflecting holistic substrate use.\nCovalent Attachment Claims\nThe manuscript now includes a statement about the possible covalent linkage of\nbacteria to electrodes via ester–amine interactions. However, the evidence remains\nindirect. I strongly recommend that the authors clearly state in the discussion or\nconclusion section that conclusive experimental evidence for covalent bonding is\nlacking. This point is essential for readers and should not be overlooked, as it currently\nappears only once in the manuscript and could easily be missed.\nResponse: We sincerely appreciate your rigorous assessment of our bonding\ncharacterization data and fully agree with your perspective. As you rightly noted, while\nour FTIR analysis (Supplementary Fig. 4) suggest possible covalent interactions\nbetween NHS ester groups and bacterial membrane amines, the evidence remains\nindirect. We unequivocally acknowledge that conclusive experimental proof of covalent\nbond formation is currently lacking.\nIn direct response to your recommendation, we now explicitly state in revision:\n\"Although spectroscopic shifts and enhanced binding stability imply ester-amine\ncovalent linkage at the biotic-abiotic interface, we emphasize that molecular-level\nconfirmation is absent. Future studies should address this mechanistic uncertainty.\"\nRevised Conclusion (Final Paragraph):\nWe added: \"Notably, while the PAA@NHS hydrogel enables robust bacterial adhesion,\nconclusive evidence for covalent bonding remains elusive and merits further\ninvestigation.\"\nWe thank you for highlighting this critical nuance—your insight significantly\nstrengthens the manuscript’s scientific rigor. The revised text will appear in track-\nchanges mode to facilitate verification.\nDepiction of Cytochrome–Electrode Binding\nSimilarly, if there is no direct evidence that cytochromes are bound to the electrode,\nthe graphical representations (e.g., “wavy bonds” in Figures 1b, 2a, and 4a) should be\nmodified to avoid overstating this claim.\nResponse: We thank the reviewer for pointing this out. As suggested, we have revised\nthe graphical illustrations in Figures 1b, 2a, and 4a to avoid overstating direct covalent\nbonding between cytochromes and the electrode surface. Specifically, the previously\nused “wavy bond” symbols—intended to imply potential bonding interactions—have\nbeen replaced or modified to reflect the current spectroscopic evidence more accurately,\nwhich indicates possible ester–amine interactions but lacks molecular-level\nconfirmation. These updates ensure the illustrations now better align with the\nexperimental findings and the cautious interpretation presented in the manuscript.\nFig. 1 | PEC-bacterial biohybrid system. (a) Schematics of the PEC-bacterial reactor for succinic acid\ngeneration; (b) schematics of interfacial connections in NiO@PAA@NHS/Au@A. succinogenes\nbiohybrid.\nFig. 2 | Visual representation of bacterial adhesion and Au NPs integration. (a) Schematic\nillustration of Au-ALE of A. succinogenes; (b) TEM image of thin-sectioned wild type A.\nsuccinogenes, the scale bar is 200 nm; (c) TEM image of thin-sectioned A. succinogenes after Au-\nALE (Au@A. succinogenes), the scale bar is 200 nm; (d) HADDF-STEM image of thin-sectioned\nAu@A. succinogenes, the scale bar is 200 nm; (e) EDS mapping image of Au in Au@A. succinogenes,\nthe scale bar is 200 nm; (f) fluorescence intensity in flow cytometry with gated events of A.\nsuccinogenes culturing in high concentration of Au ion (2 μM) and culturing in gradient ALE\nprocess (0.5 μM, 1 μM, 1.5 μM, and 2 μM); (g) schematic illustration of the interaction between\nNiO@PAA@NHS and Au@A. succinogenes bacteria; (h) SEM image of NiO/Au@A. succinogenes,\nthe scale bar is 1 μm; (i) SEM image of NiO@PAA@NHS/Au@A. succinogenes, the scale bar is 1 μm;\n(j) cellular density measurements of Au@A. succinogenes on bare NiO and on NiO@PAA@NHS,\nexpressed as cell counts per square millimeter (106/mm2), Data are represented as mean±SD (n=3).\nFig. 4 | Mechanism of succinic acid generation in biohybrid. (a) Schematic illustration of the\nextracellular-intracellular electron uptake and succinic acid production mechanism of\nNiO@PAA@NHS/Au@A. succinogenes biohybrid; (b) fluorescence lifetime spectra in A. succinogenes\nand Au@A. succinogenes; (c) fluorescence spectra of A. succinogenes before and after PEC reactions\n(A. succinogenes@Au in biohybrid was stained by DiOC (3) green membrane potential dyeing probe in\n2\nadvance, λ = 482 nm); (d) relative ATP level of after and before reaction in A. succinogenes,\nexc\nNiO@PAA@NHS/A. succinogenes, and NiO@PAA@NHS/Au@A. succinogenes; (e) NADH/NAD+ ratios\nin A. succinogenes, NiO@PAA@NHS/A. succinogenes, and NiO@PAA@NHS/Au@A. succinogenes; (f)\nrelative mRNA expression level of PK, PEPCK, FRD, PflB, and ACK in A. succinogenes,\nNiO@PAA@NHS/A. succinogenes, and NiO@PAA@NHS/Au@A. succinogenes. Data are represented\nas mean ± SD (n=3). 0.01<*p<0.05, 0.001<**p<0.01, ***p<0.001.\nFigure 2e (EDS Mapping)\nThe current size of Fig. 2e makes it difficult to interpret the EDS mapping, particularly\nfor Au within the cells. Could the authors enlarge this panel for clarity?\nResponse: We appreciate the reviewer’s suggestion. In response, we have enlarged Fig.\n2e to improve the clarity and visibility of the EDS mapping, particularly for the\ndistribution of Au within the bacterial cells. The updated figure facilitates more intuitive\ninterpretation and better highlights the intracellular localization of Au nanoparticles.\nFig. 2 | Visual representation of bacterial adhesion and Au NPs integration. (a) Schematic\nillustration of Au-ALE of A. succinogenes; (b) TEM image of thin-sectioned wild type A.\nsuccinogenes, the scale bar is 200 nm; (c) TEM image of thin-sectioned A. succinogenes after Au-\nALE (Au@A. succinogenes), the scale bar is 200 nm; (d) HADDF-STEM image of thin-sectioned\nAu@A. succinogenes, the scale bar is 200 nm; (e) EDS mapping image of Au in Au@A. succinogenes,\nthe scale bar is 200 nm; (f) fluorescence intensity in flow cytometry with gated events of A.\nsuccinogenes culturing in high concentration of Au ion (2 μM) and culturing in gradient ALE\nprocess (0.5 μM, 1 μM, 1.5 μM, and 2 μM); (g) schematic illustration of the interaction between\nNiO@PAA@NHS and Au@A. succinogenes bacteria; (h) SEM image of NiO/Au@A. succinogenes,\nthe scale bar is 1 μm; (i) SEM image of NiO@PAA@NHS/Au@A. succinogenes, the scale bar is 1 μm;\n(j) cellular density measurements of Au@A. succinogenes on bare NiO and on NiO@PAA@NHS,\nexpressed as cell counts per square millimeter (106/mm2), Data are represented as mean±SD (n=3).\nIntracellular Au Explanation\nI recommend including in the manuscript the explanation provided in the response\nregarding the presence of intracellular Au. This is important contextual information for\nthe interpretation of EDS data.\nResponse: As recommended, we have incorporated the explanation regarding the\nintracellular localization of Au nanoparticles (Au NPs) into the revised manuscript to\nenhance the interpretability of the EDS data. Specifically, we clarified that the Au NPs\nare predominantly localized near the inner membrane, a distribution that supports their\nfunction as intracellular electron mediators in our PEC-biohybrid system.\n“This membrane-associated localization of Au NPs can be attributed to their\nintracellular formation mechanism. During the ALE process, A. succinogenes\ngradually adapted to increasing concentrations of HAuCl , reducing Au3+ to Au0 using\n4\nintracellular reductants such as NADH and NADH-dependent reductases. Since many\nof these reductases are enriched at or near the inner membrane, the nucleation of Au0\npreferentially occurs in that region, resulting in the accumulation of Au NPs adjacent\nto the membrane. Although Fig. 2e shows the overall Au distribution, this proximity to\nthe membrane is further supported by HAADF-STEM and EDS mapping. Such spatial\npositioning is essential for the Au NPs to serve as intracellular conduits for electron\ntransfer, bridging photoelectrode-derived electrons with bacterial metabolic pathways\nwithout disrupting cellular viability.”\nUnchanged Figure 1\nThe authors mention they revised the depiction of covalent bonding in Fig. 1, but the\nfigure appears unchanged. Could they check if the updated version was accidentally\nomitted?\nResponse: We sincerely thank the reviewer for pointing this out. We regret that the\nupdated version of Fig. 1 was mistakenly omitted from the previous revision. We have\nnow carefully replaced it with the corrected version, in which the depiction of covalent\nbonding has been revised to avoid overstating the evidence. The updated figure has\nbeen incorporated into the revised manuscript accordingly.\nFig. 1 | PEC-bacterial biohybrid system. (a) Schematics of the PEC-bacterial reactor for succinic acid\ngeneration; (b) schematics of interfacial connections in NiO@PAA@NHS/Au@A. succinogenes\nbiohybrid.\nFTIR Interpretation and References\nFurther clarification is needed regarding the FTIR data (Figs R2–4). The authors assign\na peak at 1043 cm-1 to C–N stretching, but typical C–N stretches appear in the 1200–\n1350 cm⁻¹ range. Additionally, references are missing for several assignments (e.g.,\n1043 cm⁻¹ and 1685/1625 cm⁻¹). Please provide supporting references for all spectral\nassignments to substantiate the claims.\nResponse: We thank the reviewer for this important clarification. We have corrected\nthe spectral assignments and provided appropriate references in the revised manuscript.\nSpecifically, the peaks at 1627 cm-1 and 1533 cm-1 in A. succinogenes correspond to the\namide I and amide II bands of secondary amide groups, respectively. In the\nNiO@PAA@NHS/A. succinogenes biohybrid system, the peaks at 1685 cm-1 and 1625\ncm-1 are red-shifted relative to those in the pristine bacteria, which may reflect changes\nin hydrogen bonding or increased bond strength due to amide linkage formation R5.\nAdditionally, the band at 1043 cm-1 (and the closely related 1038 cm-1) is now\ncorrectly attributed to C–O–C stretching vibrations of polysaccharides present on the\nbacterial cell wall or C–O stretching in polyacrylic acid (PAA), rather than C–N\nstretching R6. These assignments have been updated and substantiated with appropriate\nreferences in the revised manuscript. We appreciate the reviewer’s detailed feedback,\nwhich has helped us improve the accuracy and rigor of our spectroscopic interpretation.\nSupplementary Fig. 5 – Bacterial Binding\nThe resolution of Supplementary Fig. 5 makes it difficult to assess bacterial binding.\nThe bacterial coverage also appears sparse. Could the authors explain how this image\nsupports their claim of stable binding, as stated on pages 12–13 of their manuscript?\nResponse: We thank the reviewer for this valuable comment. To evaluate the binding\nstability of A. succinogenes on the photoelectrode, we examined whether bacterial\ndetachment occurred under photoelectrochemical (PEC) conditions. Specifically,\nNiO@PAA@NHS/Au@A. succinogenes photoelectrodes were immersed in PBS for 30\nand 60 minutes, after which the surrounding PBS solution was collected and analyzed\nusing confocal laser scanning microscopy (CLSM). The bacteria were pre-stained with\nN01 fluorescent dye to enable visualization. In both cases, only faint green fluorescence\nwas detected in the PBS solution, suggesting minimal bacterial detachment. This result\nindicates that most of the bacteria remained firmly bound to the electrode surface,\nsupporting our claim of stable immobilization under PEC operating conditions.\nFigures R2–6 and H /CO Production\nshould discuss the production of H and CO during the experiments.\n2 2\nhave experimental or literature evidence that A. succinogenes can also consume CO?\nCO is typically toxic for most bacteria, with some exceptions like Clostridia.\nAdditionally, the reported CO production (~400 mmol) appears unusually high compared\nto H (~140 µmol after 8 h). What catalyst is responsible for H and CO production\n2 2\nin the system? Is there a possible error in the y-axis of the plots? The term “sealed\nsystem” also requires clarification in the context of CO production and GC\nmeasurements.\nResponse: We sincerely appreciate the reviewer's insightful question. Figure R2–6a has\nnow been included in the Supplementary Information, and we have expanded the\ndiscussion of H generation accordingly.\ncertain anaerobic bacteria such as Clostridium species, A. succinogenes lacks the\nenzymatic machinery required for CO oxidation, and CO is typically toxic to this\norganism.\nTo investigate whether gaseous byproducts (CO and H ) are formed during the\nchromatography (GC) system. In this configuration, the reactor was completely closed,\nand a concentrated bolus of CO was injected into the headspace prior to the reaction.\nstandard PEC-biohybrid conditions, where continuous CO bubbling and gas exchange\n2\nwith the atmosphere are used. Due to the artificially elevated CO partial pressure in\n2\nthe sealed system, we observed a transient enhancement in CO production, leading to\nan anomalously high measured value of 425.7 mmol after 3 hours. In contrast, H₂\nproduction under the same conditions was much lower (~140 μmol after 8 hours). These\nvalues, especially for CO, are not representative of the actual reaction environment and\nshould be considered methodological artifacts arising from the atypical sealed\nconditions. These data should therefore be interpreted as methodological artifacts\nrather than physiological outcomes. Subsequently, we implemented a headspace\nsyringe sampling protocol with GC injection, which detected negligible CO or H . This\n2\nconfirms that during authentic catalytic operation, electrons are predominantly\nchanneled into bacterial metabolism.\nPEC Stability and Long-Term Data\nFigure 3d only shows short-term (2 h) photoelectrochemical stability. Could the authors\ninclude full photocurrent traces for long-term experiments (e.g., 24 h or more) in the\nSupplementary Information?\nResponse: We thank the reviewer for highlighting this point. Since the actual catalytic\nexperiments were conducted under a 12-hour light/dark cycling strategy, we have now\nincluded additional photocurrent stability data under continuous 12-hour illumination\nat different applied voltages in the Supplementary Information. These data are intended\nto support the voltage selection process and should be interpreted as a reference to\ncomplement the succinic acid yield rate results shown in Fig. 3e.\nSupplementary Fig. 18 | Time-dependent current densities of biohybrid system at\nvarious applied potentials for 12 h.\nFurthermore, long-term photoelectrochemical performance over 120 hours was\nalready evaluated and presented in Fig. 3g. The corresponding photocurrent traces\nshow that the current remains largely stable or exhibits a slight increase over time,\nindicating the good operational stability of the PEC-biohybrid system.\nElectroactivity of A. succinogenes, mechanism of Electron Uptake and Role of H\nsuccinogenes is not electroactive and needs electron shuttles to uptake electron from\nthe electrode. The authors use strain 130Z (ATCC 55618), which corresponds to the\nwild type. If A. succinogenes could produce their own electron mediators, that could\nchallenge the claim that the system operates via direct electron transfer. Are the authors\naware of whether A. succinogenes produces endogenous electron shuttles? As a\ncomparison, Shewanella oneidensis uses flavins for mediated electron transfer despite\nhaving direct electron transfer pathways.\nResponse: We thank the reviewer for raising this important mechanistic question. We\nacknowledge that wild-type A. succinogenes (strain 130Z, ATCC 55618) is not\ntraditionally classified as electroactive, and we did not introduce any external redox\nmediators during the experiments. However, several observations support the notion\nthat electrons generated at the photoelectrode were successfully received and utilized\nby the bacteria:\n(1) Membrane potential changes were detected using the voltage-sensitive dye\nDiOC₂(3), showing a red shift in fluorescence emission after PEC operation (Fig. 4c),\nconsistent with increased intracellular electron accumulation.\n(2) Fluorescence lifetime spectroscopy revealed a 45% reduction in lifetime upon Au\nNP integration (Fig. 4b), indicating enhanced electron transfer kinetics.\n(3) Upregulation of key metabolic genes involved in succinate synthesis (e.g., PEPCK,\nFRD) was observed via RT-qPCR (Fig. 4f), suggesting metabolic activation in response\nto external electron input.\n(4) Increased ATP levels and altered NADH/NAD⁺ ratios were also observed (Fig. 4d–\ne), further supporting intracellular utilization of externally supplied electrons.\nThese results are discussed in the main text, and collectively indicate that cathodic\nelectrons influenced intracellular redox balance and energy metabolism.\nAs for the potential mechanism of electron uptake, we propose two possibilities,\nboth of which are discussed in the manuscript:\nDirect electron transfer (DET): It is plausible that membrane-bound redox proteins\nsuch as cytochrome bd oxidases (and potentially ferredoxin-like proteins involved in\noxidative phosphorylation) facilitate DET from the photoelectrode into the bacterial\nelectron transport chain.\nIndirect or hybrid mechanism: We cannot fully exclude the possibility that trace\namounts of H produced at the photoelectrode surface are utilized by membrane-\n2\nassociated hydrogenases, enabling indirect electron uptake via H oxidation and\n2\nsubsequent metabolic integration. This possibility is acknowledged and discussed in\nthe context of electron utilization pathways in the revised manuscript.\nTo our knowledge, A. succinogenes is not known to produce endogenous soluble\nelectron shuttles such as flavins, as seen in Shewanella oneidensis. Nonetheless, we\nagree that further studies (e.g., shuttle extraction assays, transcriptomics) are\nwarranted to comprehensively rule out contributions from any secreted mediators.\nWe thank the reviewer again for prompting this important mechanistic discussion,\nwhich we believe adds depth to our interpretation of the electron transfer processes in\nthe PEC-biohybrid system.\nReference 49 (Pateraki et al.) describes two possible electron uptake pathways in A.\nsuccinogenes: direct electron transfer and uptake of H₂ produced under cathodic bias.\nThe first pathway is not supported by experimental data. The latter pathway seems more\nplausible and is supported by the authors’ own findings (fig. R2–11). The argument for\ndirect electron transfer is difficult to sustain given the use of a non-electroactive strain\nand lack of strong evidence for covalent cell–electrode linkage. To confirm direct\nelectron transfer, experiments with hydrogenase knockout strains would be required,\nand strongly encouraged.\nResponse: We thank the reviewer for this important and insightful comment regarding\nthe electron uptake mechanism in Actinobacillus succinogenes 130Z. We fully agree\nthat genetic knockout of hydrogenase genes would offer the most direct method for\nconfirming whether the system operates via direct or H -mediated electron transfer.\nAsuc1277 gene (putatively encoding a membrane-bound hydrogenase), following a\nhomologous recombination approach.\nHowever, our attempts were unsuccessful due to the intrinsic genetic intractability\nof A. succinogenes 130Z. During our attempt to construct a suicide plasmid targeting\nthe Asuc1277 gene via homologous recombination (performed at Sangon Biotech), we\nobserved a strong dependence on cultivation conditions. PCR amplification of\nhomologous arms was only successful when bacterial cultures were grown in an\nanaerobic jar; cultures grown under aerobic conditions yielded no amplification. After\nstreaking both cultures, colonies displayed similar morphology, and five single clones\nfrom each condition were selected for PCR verification using internal primers for\nAsuc1277. Notably, only one clone from the anaerobic condition showed a positive\namplification band. However, upon subculturing this clone under aerobic conditions,\nthe PCR signal again disappeared. These findings suggest that although the ATCC\n55618 strain of A. succinogenes can grow under both aerobic and anaerobic conditions,\nits outer membrane structure likely undergoes significant changes depending on oxygen\navailability. Under aerobic conditions, the membrane may exhibit increased lipid\nsaturation to reduce susceptibility to oxidative stress, leading to reduced membrane\nfluidity. Alternatively, the cell envelope may adopt a Gram-positive–like architecture\nwith a thickened peptidoglycan layer, thereby hindering DNA accessibility and PCR\namplification. Moreover, efficient plasmid transfer during conjugation in Gram-\nnegative bacteria relies on the type IV secretion system (T4SS). Structural changes\nunder aerobic conditions may impair this process, thereby reducing the success of\nconjugation-based gene editing.\nWe also consulted experienced microbial genetic engineering laboratories, but all\nconfirmed that gene editing in this strain—particularly hydrogenase knockout—is\nhighly challenging and, to our knowledge, has not been successfully reported in the\nliterature. Thus, although we strongly support the rationale of using hydrogenase\nknockout strains to resolve this mechanistic question, the current technical limitations\nof A. succinogenes prevent us from implementing this strategy at this stage.\nNevertheless, we have provided multiple lines of evidence supporting electron\nuptake and metabolic integration in the biohybrid system: Fluorescence membrane\npotential shift using DiOC₂(3) (Fig. 4c), fluorescence lifetime reduction indicating\nenhanced charge separation (Fig. 4b), upregulation of metabolic genes involved in\nsuccinate biosynthesis (Fig. 4f), and changes in intracellular ATP and NADH/NAD⁺\nlevels (Fig. 4d–e).\nTherefore, we propose two plausible electron uptake routes in our system:\nDirect electron transfer (DET): Possibly mediated by cytochrome bd oxidases or\nferredoxin-like proteins in the bacterial membrane.\nIndirect or hybrid mechanism: Trace amounts of H generated at the photoelectrode\n2\ncould be oxidized by membrane-associated hydrogenases, enabling indirect electron\nentry into the metabolic network.\nWe appreciate the reviewer’s insightful recommendation, and we have updated the\nmanuscript to clearly present both mechanistic possibilities while acknowledging the\ncurrent limitations in genetic tractability of this strain.\n13C Isotopic Labelling Experiments\nI would like to commend the authors for the well-executed 13C-labelling experiments.\nThe insights into carbon flux and succinate biosynthesis are valuable and clearly\nstrengthen the manuscript.\nResponse: We sincerely thank the reviewer for their kind words and positive feedback.\nWe greatly appreciate your recognition of the 13C-labeling experiments and their\ncontribution to elucidating the carbon flux and succinate biosynthesis pathway. Your\nencouraging comments are highly motivating and have helped strengthen the overall\nquality of the manuscript.\nR1. Kim, J. H. et al. Electrochemical deprotonation of halohydrins enables cascading reactions for\nCO capture and conversion into ethylene carbonate. Nat. Commun. 16, 5038 (2025).\nsuccinogenes: a promising native succinic acid producer. Biofuels Bioprod. Biorefining 14, 950–964\n(2020).\nR3. Mukherjee, T. & Venkata Mohan, S. Bio-Electrocatalytically Regulated Selective Succinic\nAcid Production by Suppressing Pyruvate Channel Using Glycerol and CO . ACS Sustainable Chem.\nR4. Amulya, K. & Venkata Mohan, S. Augmenting succinic acid production by bioelectrochemical\nsynthesis: Influence of applied potential and CO availability. Chem. Eng. J. 411, 128377 (2021).\ngrafting of dopamine onto poly(acrylic acid). R. Soc. Open Sci 9, 211637 (2022).\nR6. Faghihzadeh, F., Anaya, N. M., Schifman, L. A. & Oyanedel-Craver, V. Fourier transform\ninfrared spectroscopy to assess molecular-level changes in microorganisms exposed to nanoparticles.\nNanotechnol. Environ. Eng. 1, 1 (2016).\nReviewer #3 (Remarks to the Author):\nThe reviewers had numerous critical questions about the article, which the authors\naddressed in detail in their response. In my opinion, most of the points were answered\nsatisfactorily and the manuscript was adapted accordingly. The manuscript can\ntherefore be published in its current form.\nResponse: We sincerely thank the reviewer for their thoughtful evaluation and positive\nrecommendation. We truly appreciate your recognition of our efforts to address the\nconcerns and improve the manuscript.\nResponse to the reviewer’s comments\nI thank the authors for addressing my comments and for their detailed responses. As a\nfollow-up to the previous round of comments, I would like to raise the following\nadditional points and questions.\nResponse: We sincerely thank the reviewer for their positive feedback and valuable\nfollow-up questions. We have provided comprehensive point-by-point responses to\neach of the additional points raised, which have further strengthened the clarity and\nvalidity of our work. The reviewer's insightful comments have been instrumental in\nhelping us improve the manuscript, and we are truly grateful for their time and\nconstructive input.\nReplacing words / correcting typos:\n1. On page 11 of the manuscript, please replace “using” with “likely using” in the\nfollowing sentence:\n“…increasing concentrations of HAuCl , reducing Au3+ to Au0 using intracellular\n4\nreductants such as NADH and NADH-dependent reductases.”\nResponse: We thank the reviewer for their careful reading and have incorporated the\nsuggested change on page 11 of the revised manuscript.\n2. Replace “HADDF” with “HAADF” in the caption of Figure 2d.\nResponse: We thank the reviewer for pointing this out. The acronym in the caption for\nFig. 2d has been updated to \"HAADF\" to ensure accuracy.\n3. On page 13, please rephrase the following sentence to avoid overstating the claim,\nas there is no conclusive evidence of covalent conjugation:\n“These results demonstrate that the covalent conjugation between the electrode and\nbacteria significantly enhances binding stability.”\nResponse: We agree with the reviewer and have rephrased the sentence on page 13 as\nsuggested to avoid overstatement. The revised text now reads: “It is likely that the NHS-\nactivated hydrogel layer could improve interfacial adhesion and biofilm stability.” We\nthank the reviewer for helping us improve the accuracy of our claim.\n4. In Figure 3h (and anywhere else in the manuscript or SI), the acronym MF is not\nexplained. Please add its meaning.\nResponse: We thank the reviewer for pointing this out. The acronym \"MF\" has now\nbeen defined as \" Microbial Fermentation\" in the caption of Fig. 3h.\n5. On page 18, replace “indicate” with “suggest” in the following sentence:\n“These results indicate that external energy input, particularly through the PEC model,\ncan substantially increase the rate of succinic acid production by A. succinogenes,\nhighlighting the potential of this approach in bioprocess optimization and sustainable\nchemical production.”\nResponse: We have replaced \"indicate\" with \"suggest\" on page 18. We appreciate the\nfeedback.\n6. In Supplementary Table 2, the fitting data is reported with excessive decimal points.\nPlease reduce this to 2–3 decimal places.\nResponse: The fitting data in Supplementary Table 2 has been updated to 2-3 decimal\nplaces. We appreciate the feedback.\nA. succinogenes A. succinogenes@Au\ny0 2.01 ± 0.11 1.02 ± 0.37\nA1 1196.24 ± 27.77 1927.63 ± 9.90\nt1 0.95 ± 0.02 0.50 ± 0.004\nA2 1423.78 ± 23.32 1094.10 ± 7.90\nt2 3.08 ± 0.05 3.10 ± 0.04\nA3 393.41 ± 8.72 111.38 ± 7.58\nt3 12.38 ± 0.15 14.22 ± 0.71\nR-Square 0.999 0.997\nt-total 3.45 ns 1.89 ns\n7. Add experimental details about the conditions used to the caption of Supplementary\nFigure 18.\nResponse: We have added the experimental details to the caption of Supplementary\nFigure 18 as follows:\n“Supplementary Fig. 18 | Time-dependent current densities of the\nNiO@PAA@NHS/Au@A. succinogenes biohybrid system measured at applied\npotentials ranging from –0.2 V to –0.5 V (vs RHE) under simulated solar illumination\n(AM 1.5G, 100 mW cm-2), using a three-electrode configuration with Pt as the counter\nelectrode and Ag/AgCl as the reference electrode.”\nIR:\nThe assignment of the IR bands is confusing. The authors correctly assigned the bands\nat 1627 and 1533 cm-1 to amide I and amide II in free bacteria. If these bands are already\npresent in the bacteria, they should also appear when the bacteria are on the electrode\n(since only a small fraction of the cell membrane contacts the electrode). However, the\nauthors report new bands at 1685 and 1625 cm-1 for the bacteria/photoelectrode system,\nsuggesting either a red-shift of the original bands due to hydrogen bonding (reasonable,\nbut it does not explain the absence of the 1533 cm-1 band) or the formation of new\namide bonds between the surface and the bacterial membrane.\nI am not fully convinced that a relatively small number of new amide bonds at the\nbacteria–electrode interface could both shift the bands and obscure the strong band at\n1533 cm-1. Have the authors considered these points? I suggest they report these\nfindings more cautiously, as the current data do not fully support their claim.\nResponse: We sincerely thank the reviewer for this insightful comment. The\ncharacteristic bands of free A. succinogenes appear at 1627 cm-1 (amide I) and 1533\ncm-1 (amide II), consistent with literature reports. Upon integration with the\nNiO@PAA@NHS electrode, the biohybrid exhibits two partially shifted features at\n1685 cm-1 and 1625 cm-1, accompanied by a marked reduction in the amide II intensity.\nThese spectral changes are primarily attributed to strengthened hydrogen bonding and\ninterfacial interactions between bacterial surface proteins and the NHS-activated\nhydrogel layer.\nWe acknowledge that only a small fraction of the bacterial membrane directly\ncontacts the electrode; therefore, complete disappearance of the 1533 cm-1 band is\nunlikely. The apparent attenuation of this band may result from spectral convolution\nwith the strong C=O stretching vibration of the NHS-modified PAA hydrogel and\noverlap with the broad O-H bending region around 1550-1650 cm-1.\nWhile the red-shifted amide I band (to 1685 cm-1) may suggest enhanced hydrogen\nbonding or partial amide-linkage formation at the interface, the current evidence does\nnot conclusively prove covalent bond formation. Accordingly, we have revised the\nmanuscript to report these findings more cautiously, describing them as “possible\nhydrogen-bonding and amide-linkage interactions” rather than definitive covalent\nconjugation. Corresponding revisions have been made in the main text to ensure an\naccurate and balanced interpretation.\nSEM:\nFor Figures 2h and 2i, can the authors provide SEM images of the photocathodes with\nbacteria at a wider field of view than 200 nm? This would help readers appreciate the\ndensity of the biofilms formed.\nResponse: We thank the reviewer for raising this point regarding the SEM field of view.\nWe agree that visualizing the biofilm density is important. To that end, the images in\nFigures 2h and 2i were already captured at a wider field of view (1 μm scale bar) to\nfully capture the biofilm architecture and cell distribution. The higher-magnification\nimages (200 nm) in Figures 2b–2e were included specifically to resolve the\nultrastructural details, such as the intracellular Au nanoparticles. We believe the\nprovided images collectively offer both an overview of the biofilm and a detailed look\nat the key nanoscale features.\nBiofilm stability:\nRegarding the discussion on page 13 of the confocal laser scanning microscope analysis:\nhave the authors performed exclusion controls using bare photoelectrodes (i.e., NiO\nand NiO/PAA without NHS) to study biofilm stability in comparison with the final\nphotoelectrodes? Such control experiments would strengthen the results and\nconclusions.\nResponse: We thank the reviewer for this valuable suggestion. Following the reviewer’s\nadvice, we have now performed additional control experiments using bare NiO and\nNiO@PAA electrodes to evaluate biofilm stability in comparison with the final\nNiO@PAA@NHS photoelectrode. As shown in Fig. R3 (Supplementary Fig. S5 in\nSupplementary Information of new version), confocal laser scanning microscopy\n(CLSM) was used to visualize bacteria detached into the surrounding PBS solution after\n30 min of immersion.\nFig. R4 CLSM images of A. succinogenes bound on (a) NiO@PAA@NHS, (b)\nNiO@PAA, and (c) NiO electrodes after 30 min PBS immersion, showing minimal\ndetachment on the NHS-activated surface.\nThe NiO@PAA@NHS electrode (Fig. R4a) exhibited almost no fluorescent signal\nin the supernatant, indicating that only a few A. succinogenes cells were released,\nwhereas the NiO@PAA (Fig. R4b) and bare NiO (Fig. R4c) electrodes showed\nprogressively stronger fluorescence signals corresponding to higher bacterial\ndetachment. These results clearly confirm that the NHS-activated hydrogel layer\ngreatly improves the interfacial adhesion and biofilm stability of the immobilized\nbacteria. The corresponding description has been added to the revised manuscript, and\nthe new CLSM images have been included as Supplementary Fig. 5.\nHydrogen evolution:\nOn page 19, the authors state: “At -0.2 V (vs RHE), the photocurrent density is lower,\nsuggesting that the electron supply to the biohybrid system is insufficient to meet the\nbacteria's needs for succinic acid production (Supplementary Fig. 18). This could be\ndue to less efficient charge separation or a lower driving force for electron transfer at\nthis potential.” And “Conversely, when the potential is more negative, such as at -0.4 V\nand -0.5 V, the photocurrent density increases, indicating an increased supply of\nelectrons.” At -0.2 V, the photocurrent density is already ~1 mA/cm², which is\nsignificant and likely generates considerable H , since no succinic acid is being\n2\nproduced. At -0.4/–0.5 V, the current increases to ~6 mA/cm2, which would produce\neven more H . Electron transfer across the cell membrane is almost certainly the rate-\n2\nlimiting step, so charges from the photoelectrode are likely being consumed for H\n2\nevolution, as suggested by Supplementary Fig. 19a.\nHave the authors performed exclusion controls at -0.2, -0.3, -0.4, and -0.5 V vs\nRHE without bacteria present (in batch and under flow conditions)? It seems very likely\nthat hydrogen evolution occurs under all of these conditions. These experiments are\ncritical, especially given the data in Supplementary Fig. 19a showing that the biohybrid\nsystem produces 140 μmol of H at -0.3 V. If not yet performed, such controls are\n2\nessential to confirm whether bacteria are directly accepting electrons or instead using\nH as an indirect mediator.\nmechanism behind the increased succinic acid production. To confirm that the\nenhancement is primarily due to direct electron transfer (DET), we conducted the\nfollowing experiments:\n(1) Enhanced electroactivity of A. succinogenes after Au domestication.\nAs shown in the newly added Fig. R1 (Supplementary Fig. 25a in Supplementary\nInformation of new version), the CV profiles of wild-type A. succinogenes (black curve)\nexhibit only a weak redox response, indicating inherently poor electroactivity and\nlimited ability to exchange electrons with the electrode. This observation is consistent\nwith the established understanding that wild-type A. succinogenes is a non-model\nelectrotroph with a limited native capacity for extracellular electron uptake. In contrast,\nafter Au adaptive laboratory evolution, the Au@A. succinogenes cells (red curve)\ndisplay a pronounced increase in both anodic and cathodic current densities, with a\nclear pair of redox peaks centered at approximately –0.45 V (vs Ag/AgCl). The current\ndensity nearly doubled from ~0.45 mA cm-2 to ~0.85 mA cm-2, confirming that Au\nnanoparticle domestication significantly improves the bacterial redox activity. This\nenhancement arises because Au nanoparticles, formed near the inner membrane,\nprovide highly conductive nanoscale bridges that facilitate charge exchange between\nthe electrode surface and intracellular redox proteins (e.g., cytochrome bd complexes\nor ferredoxin-like proteins).\nFig. R1 Cyclic voltammetry (CV) curves of wild-type A. succinogenes (black) and Au-\ndomesticated A. succinogenes (red).\nTo exclude the influence of any soluble redox mediators, we further performed CV\nmeasurements on the cell-free supernatant obtained after centrifugation of Au-\ndomesticated A. succinogenes. As shown in Fig. R2 (Supplementary Fig. 25b in\nSupplementary Information of new version), the supernatant (black line) exhibits nearly\nfeatureless current responses, whereas the Au@A. succinogenes cells (red line) show\ndistinct redox peaks. This clearly indicates that the electrochemical activity originates\nfrom the bacteria themselves rather than from any residual Au ions or soluble mediators\nin the culture medium. Moreover, all bacterial samples for CV analysis were carefully\npurified: the cells were harvested by centrifugation and washed three times with\nphosphate-buffered saline (PBS) before measurement to remove extracellular residues\nand ensure that the signals reflect intrinsic bacterial electroactivity. The CV scans were\nconducted at 5 mV s-1, which are appropriate for biological systems, minimizing\ncapacitive artifacts while maintaining adequate redox resolution.\nFig. R2 Cyclic voltammetry (CV) profiles of Au@A. succinogenes (red) and the\ncorresponding cell-free supernatant (black).\nThese CV results are also consistent with the reduced fluorescence lifetime (Fig.\n4b) and the red-shift of the DiOC2(3) probe (Fig. 4c), collectively supporting\naccelerated transmembrane electron flow after Au incorporation.\n(2) Control experiment excluding H -mediated reduction.\n2\nintroduced 400 mL h-1 of H into the reactor, a rate far exceeding the amount that could\n2\nbe generated electrochemically by the photocathode. For comparison, the\nphotocathode itself produced only ~259.9 μmol of H within 8 h, equivalent to ~32.5\n2\nμmol h-1 or approximately 0.8 mL h-1 under standard conditions. Therefore, the\nexternally supplied H flux was roughly 500 times higher than the electrode’s intrinsic\nmodest 18.3% increase in succinic acid yield, and the PEC-biohybrid configuration\nshowed a negligible 3.8% enhancement.\nFig. R3 Comparison of succinic acid yield rates of PEC-biohybrid and biohybrid\nsystems with and without external H supplementation.\nidentical conditions achieved a succinic acid yield rate of 1.41 g L-1 h-1 cm-2, which is\nsignificantly higher than that of the biohybrid system (0.21 g L-1 h-1 cm-2). This striking\ndifference indicates that the minor improvement under excess external H and the\n2\npronounced enhancement observed in the Au-integrated PEC-biohybrid originate from\ndistinct mechanisms. The negligible contribution of H confirms that electrochemically\n2\ngenerated H plays only a marginal role, whereas the dominant enhancement originates\n2\nfrom the engineered NiO@PAA@NHS/Au@A. succinogenes interface. This\narchitecture establishes a continuous conductive network bridging the photoelectrode\nand the bacterial inner membrane, thereby enabling rapid and efficient direct electron\ntransfer across the bio–inorganic boundary instead of relying on an indirect, H -\n2\nmediated pathway.\n(3) Following the reviewer’s suggestion, we further quantified H evolution from the\n2\nbare NiO@PAA@NHS photocathode (without bacteria) at applied potentials of –0.2, –\n0.3, –0.4, and –0.5 V vs RHE under identical illumination and electrolyte conditions.\nAs shown in Fig. R5 (Supplementary Fig. 19 in Supplementary Information of new\nversion), the accumulated H amount increased progressively from 127.7 μmol (≈ 2.86\n2\nmL, –0.2 V) to 259.9 μmol (≈ 5.82 mL, –0.3 V), 458.4 μmol (≈ 10.27 mL, –0.4 V), and\n603.6 μmol (≈ 13.53 mL, –0.5 V) after 8 h of photoelectrocatalysis. These results\nconfirm that H evolution becomes increasingly significant at more negative potentials,\n2\nconsistent with the rise in photocurrent density.\nFig. R5 Time-dependent H evolution from the bare NiO@PAA@NHS photocathode\n2\nunder different applied potentials (–0.2, –0.3, –0.4, -0.5V vs. RHE) for 8 h of\nphotoelectrocatalysis.\nHowever, the maximum succinic acid yield was obtained at –0.3 V, rather than at\nmore negative potentials. This observation indicates that although the total electron\nflux increases at –0.4 V and –0.5 V, a greater proportion of these electrons is consumed\nin H evolution rather than being transferred to the bacteria for CO fixation. Therefore,\n2 2\nH generation and transmembrane electron transfer are competitive processes. At\n2\nmoderately negative potential (–0.3 V), the interfacial charge transfer and microbial\nelectron uptake are optimally balanced, enabling the highest succinic acid productivity.\nIn contrast, at excessively negative potentials (≤ –0.4 V), accelerated H evolution\n2\ndominates electron consumption, thereby diminishing the efficiency of the biohybrid\nsystem.\nThis potential-dependent analysis further confirms that the enhanced succinic acid\nproduction originates primarily from direct electron transfer across the engineered\nNiO@PAA@NHS/Au@A. succinogenes interface, rather than from an indirect, H -\n2\nmediated pathway.\nCO/GC analysis (page 20):\nThe authors report that in a sealed reactor with bolus CO injection, substantial CO\n2\n(~425.7 mmol after 3 h) and trace H (~140 μmol after 8 h) were detected. They attribute\n2\nthis to “artificial accumulation under non-standard conditions.”\n• What exactly do the authors mean by “artificial accumulation”? If CO is not produced\nby CO reduction at the electrode, what is its origin?\n2\n• Under flow conditions, the authors claim that no CO or H was detected. This seems\n2\nunlikely; more plausibly, CO and H were flushed out by the continuous CO stream\n2 2\nand went undetected. Can the authors provide the raw GC data from these experiments?\n• It is concerning that such a large amount of CO is reported without proper justification.\nIn my previous review, I asked about this but the response suggested it was unnecessary\nto consider. Based on the reported data (2 mA/cm2 at -0.3 V for 12 h with a 5 cm2\nelectrode), I estimate a faradaic efficiency above 200% for CO, which is clearly\nproblematic. Could there be errors in GC calibration? Are the authors confident in these\nCO measurements?\nResponse: We sincerely thank the reviewer for carefully identifying this issue. After re-\nexamining our gas chromatography (GC) data and calibration files, we found that the\nunit for CO yield in the sealed reactor was mistakenly written as “mmol” instead of\n“nmol” The correct value should be 425.7 nmol of CO after 3 h, not 425.7 mmol. We\ndeeply regret this typographical error and appreciate the reviewer’s attention, which\nallowed us to identify and correct it. To ensure transparency, we have now provided the\nraw GC chromatograms, calibration curves, and quantification files for CO\nFig. R6 CO calibration curve by gas chromatography.\nFig. R7 Linear calibration curve for the quantification of CO using gas\nchromatography\nFig. R8 Gas chromatograms of CO production at different time points.\nMechanism:\nOn page 24, the authors note: “an indirect or hybrid mechanism may operate in\nparallel … Small amounts of H generated at the photoelectrode surface could be\n2\noxidized by membrane-bound hydrogenases.” I strongly support this hypothesis. Based\non the presented data, the indirect pathway is very likely and may be the dominant\nmechanism for electron uptake. The authors should explicitly acknowledge this in their\nconclusions and, if space permits, also in the abstract. On the same page, please\nrephrase the following speculative statement: “This internal conductive network not\nonly allows for electron transfer through cytochromes Cyt d I and Cyt d II but also plays\na role in the synthesis of additional redox couples (such as NADH/NAD+) and the\nenergy currency (ATP) necessary for the highly efficient synthesis of succinic acid.”\nThe evidence is insufficient to support this claim.\nResponse: We sincerely thank the reviewer for these constructive suggestions. Based\non the additional control experiments, including cyclic voltammetry, hydrogen-\nsupplementation tests, and quantification of H evolution from bare photocathodes,\n2\nwe have suggested that direct electron transfer (DET) through the\nNiO@PAA@NHS/Au@A. succinogenes interface is the predominant pathway for CO\n2\nreduction, while H -mediated electron delivery serves only as a minor auxiliary process.\nspeculative statement in revision.\nFigure 4a:\n• The schematic lacks clarity on electron transfer across the membrane. No pathway,\nprotein, or molecular shuttle is indicated. Please add an arrow with a question mark,\nand also include H at the photocathode surface, with an arrow entering the cell and\n2\nreacting with periplasmic hydrogenases and electrons being transferred to other native\nelectron mediators. Otherwise, the left-hand portion of the schematic adds little\nmechanistic value.\n• The periplasm is incorrectly labeled. It is the aqueous compartment between the inner\nand outer membranes of Gram-negative bacteria, not the hydrophobic core of the outer\nmembrane. If the authors are unsure how to correct this, they should consider removing\nthe cellular schematic altogether.\nResponse: We thank the reviewer for these critical suggestions to improve the\nmechanistic clarity of our schematic (Fig. 4a). In response, we have implemented the\nfollowing revisions: To illustrate electron transfer across the membrane, we have added\na clearly labeled arrow indicating direct electron transfer (DET) as the predominant\nmechanism. The wrong labeling of periplasm has been deleted. We believe these\nrevisions have significantly enhanced the schematic's accuracy and mechanistic value,\nand we thank the reviewer for the insightful input.\nCharge accumulation (page 26):\nThe use of DiOC to probe charge accumulation inside the bacterial membrane is\n2\ninteresting, but how have the authors controlled for the presence of sugar? Since\nglucose is present in their biohybrid system, fermentation and H oxidation could also\n2\nproduce similar red shifts. Have the authors run exclusion controls with cultures grown\nheterotrophically (i.e., glucose only, no electrode or applied potential)? Did they\nobserve similar DiOC shifts under those conditions? If so, then charge accumulation\n2\nmay not be attributable solely to photoelectrons.\nResponse: To address the reviewer’s concern, we performed an additional\nheterotrophic control using A. succinogenes cultured with glucose only, in the absence\nof any electrode or applied bias. As shown in Fig. R9 (Supplementary Fig. 27 in\nSupplementary Information of new version), the fluorescence spectra of DiOC (3)-\n2\nstained cells before and after glucose addition (black: initial; red: glucose only) are\nnearly identical, indicating that glucose metabolism alone does not cause a detectable\nred shift or fluorescence enhancement. Therefore, the membrane potential change\nobserved under photoelectrocatalytic conditions arises predominantly from electrode-\ndriven charge accumulation rather than from fermentative activity.\nFig. R9 Fluorescence spectra of A. succinogenes under heterotrophic conditions with\nglucose only (no electrode or bias).\nIn the following paragraph (Fig. 4d):\n• How were these cultures produced?\n• Specifically, how was the first dataset (A. succinogenes alone) obtained? Was there\nan electrode present, or was glucose the only carbon source? Please provide these\ndetails in the experimental section.\nThe statement: “ATP content in A. succinogenes increased by 21% upon complexation\nwith NiO@PAA@NHS …” should be revised. The word “complexation” implies\nlinkage, but there is no strong evidence for covalent bonding. Please replace with\n“biofilm formation”. More broadly, speculative terms should be avoided where\nevidence is lacking.\nResponse: We thank the reviewer for this important question. All cultures shown in Fig.\n4d were produced under identical fermentation conditions using the same fermentation\nbroth. Specifically, A. succinogenes cells were cultivated anaerobically in the standard\nfermentation medium containing glucose. For the control group (“A. succinogenes\nalone”), the bacteria were incubated in the fermentation broth without any\nphotoelectrode or applied potential. For the biohybrid systems, the NiO@PAA@NHS-\nbased photocathodes coated with A. succinogenes were immersed in the same\nfermentation broth and operated under photoelectrocatalytic conditions for the same\nduration. This ensured that any differences observed originated from the presence of\nthe photoelectrode and not from variations in culture conditions.\nWe thank the reviewer for this precise correction. The term \"complexation\" has\nbeen replaced with the more accurate description \"biofilm formation\" as suggested. We\nacknowledge that the original wording could imply a specific type of bonding not\nconclusively demonstrated. We have further reviewed the manuscript to ensure that our\nlanguage remains strictly aligned with the experimental evidence throughout.\nMethods:\nFor CO conversion efficiency, please include faradaic efficiency calculations for HER\n2\nand CO RR. Also clarify how mol/min rates were converted to absolute CO input\n2 2\nmoles — this step is missing.\nResponse: We appreciate the reviewer’s valuable comments on the Faradaic efficiency\nand CO conversion calculations. In the revised manuscript, explicit Faradaic\n2\nefficiency values for the hydrogen evolution reaction (HER, 48.3%) and for CO\nformation from CO reduction (0.38%) have been added. These calculations were based\n2\non the total charge passed (Q, C) and the quantified amount of gaseous or liquid\nproducts (n, mol) using the standard formula: FE = n × z × F ÷ Q × 100%, where\nz = 2 for both H and CO, and F = 96485 C mol-1. Regarding CO conversion efficiency,\n2 2\nwe clarified in the Methods section that the total CO input was determined by\n2\nintegrating the inlet flow over time. At a fixed CO₂ flow rate of 7 mL min-1 (298 K, 1\natm), the molar feed rate is calculated as: ṅ = 7 mL min-1 ÷ 24450 mL mol-\n(CO2, input)\n1×t(min) = 2.86 × 10-4t mol. This correction has been incorporated to ensure\ntransparency and reproducibility of the calculation.\nAdditional clarification:\nIn one response, the authors wrote: “Alternatively, the cell envelope may adopt a Gram-\npositive–like architecture with a thickened peptidoglycan layer.” Can the authors\nexplain how a Gram-negative bacterium could adopt such an architecture?\nResponse: We fully acknowledge that Actinobacillus succinogenes (ATCC 55618) is\ntaxonomically a Gram-negative bacterium, and that no experimental evidence supports\nan actual conversion to a Gram-positive cell-wall type, such as the loss of the outer\nmembrane and the formation of an extensively thick peptidoglycan layer. Our intention\nwas to describe an adaptive structural response rather than a taxonomic\ntransformation. Under certain environmental stresses, such as prolonged oxygen\nexposure, oxidative stress, or membrane-destabilizing conditions, Gram-negative\nbacteria are known to activate envelope stress responses that remodel their cell\nenvelopes. For instance, Mitchell and Silhavy (2019) reported that envelope stress can\nincrease peptidoglycan content and decrease membrane fluidity 1. In addition, other\nstudies have shown that oxidative or environmental stress can alter outer-membrane\nlipid composition by increasing saturated fatty acid content 2, thereby reducing\nmembrane fluidity and diffusivity. These findings collectively indicate that the typical\n“thin peptidoglycan plus distinct outer membrane” architecture of Gram-negative\nbacteria can become more compact, with a relatively thicker peptidoglycan layer and\na less fluid outer membrane.\nIn our study, although we did not directly measure peptidoglycan thickness or\nouter-membrane integrity, we observed that genetic manipulations, such as conjugation\nand homologous recombination, were successful only under strictly anaerobic\nconditions but failed under identical aerobic conditions. We therefore infer that aerobic\ncultivation may induce a more rigid and less permeable envelope, consistent with the\nreported stress-induced increase in lipid saturation and peptidoglycan density, which\ncould impede DNA uptake or conjugative transfer.\nThus, the phrase “Gram-positive–like architecture” was intended to describe a\nfunctional resemblance, that is, a denser and less permeable envelope, rather than a\nliteral transition to a Gram-positive cell-wall structure. To avoid misunderstanding, we\nhave revised the manuscript to specify that this represents a proposed adaptive envelope\nremodeling rather than a demonstrated structural conversion.\nWe hope this clarification addresses the reviewer’s concern and improves the\nprecision and clarity of our manuscript.\n1. Mitchell, A. M. & Silhavy, T. J. Envelope stress responses: balancing damage repair\nand toxicity. Nat. Rev. Microbiol. 17, 417–428 (2019).\n2. Bisht, R., Charlesworth, P. D., Sperandeo, P. & Polissi, A. Breaking Barriers:\nExploiting Envelope Biogenesis and Stress Responses to Develop Novel Antimicrobial\nStrategies in Gram-Negative Bacteria. Pathogens 13, 889 (2024).\nResponse to reviewer’s comments for Manuscript (number: NCOMMS-25-14204C-Z)\nWe sincerely thank Reviewer for the thorough, constructive, and insightful\ncomments. We appreciate the reviewer’s careful evaluation of both the experimental\nrigor and the clarity of mechanistic interpretation. In response, we have revised the\nmanuscript extensively to improve precision, transparency, and consistency. All\ncomments have been carefully addressed as detailed below.\nPage 13: Replace “bacteria-bound” with “bacteria with”.\nResponse: We thank the reviewer for pointing this out. The wording has been corrected,\nand “bacteria-bound” has been replaced with “bacteria with” on page 13 to improve\nclarity and accuracy.\nSupplementary Fig. 25: Convert all potentials reported vs Ag/AgCl to RHE for\nconsistency with the rest of the manuscript. This should include any mention of\nAg/AgCl on page 23 and elsewhere.\nResponse: We agree with the reviewer. All electrochemical potentials in Supplementary\nFig. 25, as well as all other instances throughout the manuscript (including page 23\nand elsewhere), have now been consistently converted and reported versus the\nreversible hydrogen electrode (RHE). Corresponding figure labels, captions, and text\nhave been revised accordingly.\nPage 23: The statement “Based on the additional electrochemical and control\nexperiments, we confirmed that direct electron transfer (DET) across the Au-mediated\nelectrode–bacteria interface serves as the dominant pathway, whereas H -mediated\n2\nelectron transfer plays only a minor auxiliary role” is very strong and does not appear\nto be equally supported by a sufficiently detailed description of the methodology. Please\nprovide the experimental conditions for Supplementary Figs. 25 and 26 (e.g.,\nelectrolyte/media composition, pH, temperature, gas atmosphere, cell density/OD,\nelectrode material and area, scan rate settings, number of replicates or scans, and how\nDET vs H contributions were quantified).\nwas overly strong. Accordingly, we have softened the mechanistic language to avoid\noverstatement. The revised text now states that the data suggest or indicate a greater\ninvolvement of direct electron transfer (DET) under the studied conditions, rather than\nclaiming definitive dominance.\nIn addition, we have substantially expanded the Methods section to provide full\nexperimental details for Supplementary Figs. 25 and 26, including electrolyte/media\ncomposition, pH, temperature, gas atmosphere, cell density, electrode material and\narea, scan rate, number of replicates, and applied potentials. The added\nmethodological descriptions are summarized below and included in the revised\nmanuscript.\nPage 24: Replace “was featureless” with “was mostly featureless”, and remove “highly”\nfrom “highly conductive”.\nResponse: We agree and have implemented these wording changes on page 24 to ensure\nmore accurate and conservative descriptions.\nPage 24: Replace “negligible” with “small”, and replace “small amounts” with “µmol\namounts”.\nResponse: We thank the reviewer for this suggestion. The requested wording changes\nhave been made to provide a more quantitative and precise description.\nSupplementary Fig. 26: What potential was applied in these experiments? More\nexperimental detail is needed. As written, it is difficult to interpret—and therefore to\nassess—the strength of several conclusions, and future readers will face the same\nissue.\nResponse: We agree that this information is essential. The applied potential (−0.3 V vs\nRHE for the PEC-biohybrid system) and the operating conditions for both PEC-\nbiohybrid and biohybrid controls have now been explicitly stated in the Methods section,\nas detailed above.\nDiOC2(3): DiOC2(3) does not allow one to conclude that “this energy comes from\nEET.” In Fig. S27, DiOC2(3) would be expected to shift with sugar consumption\nbecause metabolism can change membrane potential; similar behaviour has been\nreported previously for Shewanella oneidensis (see available literature). The reported\nresult would be more consistent with starved cells (for example, no sugar present). In\naddition, dissolved O and/or other oxidants/reductants in the medium can strongly\n2\naffect membrane potential. Because the manuscript does not provide sufficient\nexperimental details for the DiOC2(3) assays (media composition, aeration/oxygen\ncontrol, carbon source and concentration, incubation time, cell density, staining\nprotocol, instrument settings, gating/analysis, controls), it is not possible to recommend\nappropriate controls or evaluate the conclusions. I recommend removing the DiOC2(3)\nsection and associated figures, as it is not currently strong or conclusive, and specialists\nin the field are likely to identify these weaknesses fairly easily.\nResponse: We thank the reviewer for this critical and well-reasoned assessment. We\nagree that the DiOC2(3) results alone do not allow a definitive conclusion regarding\nenergy input via extracellular electron transfer (EET), particularly given the known\ninfluences of metabolism, dissolved oxygen, and redox-active species on membrane\npotential.\nIn light of these considerations, and to avoid overinterpretation, we have removed\nthe DiOC2(3) section and the associated figure from the revised manuscript.\nFig. 4a schematic: This is not a scientifically accurate representation of the Gram-\nnegative envelope of A. succinogenes. At minimum, the schematic should depict the\nouter membrane, periplasm, and inner membrane. The text repeatedly states that Au\nparticles are near the inner membrane, but the current illustration could be interpreted\nas the outer membrane, which will confuse readers. This should be corrected. In\naddition, even if the H -mediated pathway is considered minor, it should be represented\n2\n(for example, including hydrogenases and H₂ oxidation feeding electrons into cellular\nmetabolism). If the full mechanism is too complex to depict clearly, I suggest either\nsimplifying the main-text schematic to show only the glucose metabolic pathway\n(without membranes) or removing the schematic from the main text.\nResponse: We appreciate this important comment. We agree that the previous schematic\ncould be misleading with respect to the Gram-negative envelope architecture.\nFollowing the reviewer’s suggestion, we have simplified Fig. 4a to depict only the\ncentral glucose metabolic pathway leading to succinic acid production, without\nexplicitly illustrating membrane structures or detailed electron transport routes. This\nrevision improves clarity and avoids potential misinterpretation while remaining\nconsistent with the scope of the experimental evidence.\nOnce again, we sincerely thank Reviewer 2 for the detailed and constructive\nfeedback. The manuscript has been significantly strengthened as a result of these\nrevisions, and we believe that the changes improve both scientific rigor and clarity for\nfuture readers."}, {"round": 2, "reviewer_comments": {"reviewer_1": "No more questions.", "reviewer_2": "I thank the authors for their detailed responses. As a follow-up to the previous round of comments", "reviewer_questions": "nCO₂ Conversion Efficiency\nThe method used to calculate the CO₂ conversion efficiency is not described. Does this calculation account for the total amount of CO₂ present in the system at the start of the experiment? Please clarify how this metric was derived.\nReporting of Succinate Concentration\nCould the authors report the molarity of succinate produced in addition to the current units (g/L/h)? This would aid in comparison with other systems. Additionally, including the volume of medium used during the experiments would help readers better appreciate the scale of the reactor and the biohybrid system.\nFaradaic Efficiency (FE)\nWhile I understand the challenges in determining Faradaic efficiency, it remains a critical metric in evaluating (photo)electrochemical systems, including biohybrids. Could the authors report the FE for CO₂-to-succinate conversion and provide a justification for any FE values exceeding 100%? The authors could use their response.\nCovalent Attachment Claims\nThe manuscript now includes a statement about the possible covalent linkage of bacteria to electrodes via ester–amine interactions. However, the evidence remains indirect. I strongly recommend that the authors clearly state in the discussion or conclusion section that conclusive experimental evidence for covalent bonding is lacking. This point is essential for readers and should not be overlooked, as it currently appears only once in the manuscript and could easily be missed.\nDepiction of Cytochrome–Electrode Binding\nSimilarly, if there is no direct evidence that cytochromes are bound to the electrode, the graphical representations (e.g., “wavy bonds” in Figures 1b, 2a, and 4a) should be modified to avoid overstating this claim.\nFigure 2e (EDS Mapping)\nThe current size of Fig. 2e makes it difficult to interpret the EDS mapping, particularly for Au within the cells. Could the authors enlarge this panel for clarity?\nIntracellular Au Explanation\nI recommend including in the manuscript the explanation provided in the response regarding the presence of intracellular Au. This is important contextual information for the interpretation of EDS data.\nUnchanged Figure 1\nThe authors mention they revised the depiction of covalent bonding in Fig. 1, but the figure appears unchanged. Could they check if the updated version was accidentally omitted?\nFTIR Interpretation and References\nFurther clarification is needed regarding the FTIR data (Figs R2–4). The authors assign a peak at 1043 cm⁻¹ to C–N stretching, but typical C–N stretches appear in the 1200–1350 cm⁻¹ range. Additionally, references are missing for several assignments (e.g., 1043 cm⁻¹ and 1685/1625 cm⁻"}, "author_response": ""}]

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