Enhancing Solar-to-Chemical Conversion through Tailoring Dimensions of Semiconductors in Biohybrid Systems

Enhancing Solar-to-Chemical Conversion through Tailoring Dimensions of Semiconductors in Biohybrid Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhancing Solar-to-Chemical Conversion through Tailoring Dimensions of Semiconductors in Biohybrid Systems Mingming Guo*, Xinke Kong*, Wenbo Cheng, Wenjun Yang, Shanshan Pi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4431666/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Integrating light-harvesting semiconductor materials with biocatalysts offers a promising approach for solar-power production of fuels and fine chemicals. Despite significant advances, the influence of materials’ dimensions on energy utilization efficiency and the involved photoelectron transfer pathways remains largely to be explored. Here, we investigated the effect of dimensionality on the energy conversion efficiency in semiconductor nanomaterial-based biohybrid systems. We found that the intracellularly localized 2D nanoplatelets, particularly with core-crown heterostructures, were more efficient in supplying energy for microbial chemical production than the lower-dimensional nanomaterials. The biohybrids possessing the 2D nanoplatelets exhibited a 2.69-fold increase in 2,3-butanediol (BDO) production yield and achieved 2.35% solar-to-chemical conversion efficiency. Based on metabolomic and transcriptomic analyses, we identified a novel thiamine pyrophosphate (TPP)-mediated pathway of energy generation from photoexcited electrons. Furthermore, the addition of TPP enhanced the BDO production of the biohybrids under illumination. Our results demonstrate the potential to increase the solar-to-chemical conversion efficiency of semiconductor biohybrids by tailoring the dimension of semiconductor nanomaterials and engineering the intracellular electron transfer and energy generation pathways. * Mingming Guo and Xinke Kong contributed equally to this work. Biological sciences/Biotechnology/Metabolic engineering Biological sciences/Biochemistry/Biocatalysis Physical sciences/Materials science/Materials for energy and catalysis/Photocatalysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Many chemicals derived from fossil fuels could be sustainably produced via biomanufacturing, reducing the reliance on fossil energy and lowering greenhouse gas emission 1, 2 . However, current biomanufacturing processes primarily reliant on sugar feedstocks suffer from low energy conversion efficiencies and productivities, particularly in the production of highly reduced chemicals 3, 4 . Through the catabolic process, the microbial cell factories release energy nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and adenosine triphosphate (ATP) from sugar, simultaneously generating CO 2 which reduces carbon yields and represents a significant bottleneck in chemical production 5 . Semiconductor-microbe hybrid systems have emerged as a promising solution, integrating efficient light-harvesting nanomaterials with superior whole-cell biocatalysts, allowing the non-photosynthetic bacteria to directly utilize light energy to power the cells 6, 7 . These systems achieve higher carbon yield and productivities by providing additional energy while reducing or eliminating carbon loss 8 . Low-dimensional nanomaterials 9 , typically smaller than 100 nm, including zero-dimensional (0D) nanoparticles or quantum dots (QDs), one-dimensional (1D) nanotubes or nanorods (NRs), and two-dimensional (2D) nanosheets or nanoplatelets (NPLs), have been recognized for their cost-effectiveness, enhanced biocompatibility, photocatalytic activity, and stability 10-13 . These nanomaterials dominate in contemporary semiconductor biohybrid systems 14, 15 . To fully harness the potential of those low-dimensional biohybrid systems, it’s essential to engineer semiconductors and bacteria based on detailed, systemic, and quantitative insights that enhance energy conversion efficiencies 16 . Although research has begun to explore the variables influencing the effectiveness of semiconductor biohybrids, a comprehensive assessment of nanomaterial dimensions and electron transfer mechanisms is still in its infancy 17, 18 . Current studies have primarily focused on photoelectron transfer mechanisms in biohybrids with extracellular or periplasmic nanomaterial localization 19-21 , where electrons must traverse cellular membranes-an energy-consuming bottleneck 22 . Intracellular nanomaterials, however, allow for the direct generation of photoelectrons within the cytoplasm, facilitating rapid charge transfer 23 . Studies in mammalian and plant cells have emphasized the importance of the dimensional properties for nanomaterials-cell interactions, especially for biomolecule delivery, showing that size and shape can affect cellular internalization and efficiency 24-26 . Yet, the understanding of how nanomaterial dimensions and intracellular photoelectron transfer mechanisms affect solar-driven chemical production in biohybrid systems remains limited, restricting the advancement of efficient solar-driven biomanufacturing. Here we constructed and quantitatively assessed the biohybrid system’s performance, focusing on the influence of nanomaterial dimensions on energy conversion efficiency and electron transfer mechanisms. Using low-dimensional cadmium sulfide (CdS) semiconductors (0D QDs, 1D NRs, and 2D NPLs) and 2,3-butanediol (BDO)-producing engineered bacteria Vibrio natriegens , we demonstrated that 2D NPLs (5 monolayers (ML)) exhibit higher internalization rates and photocurrent, leading to increased BDO production (Fig. 1a). To enhance the charge separation efficiency of 5 ML 2D NPLs, we further designed a core/crown (CC) heterostructure. In particular, the CC NPLs structured 2D NPLs biohybrid systems notably enhanced BDO yield by 2.69 times compared to the pure bacterial systems, with a solar-to-chemical efficiency of 2.35%. Electron microscopy and photophysical analysis elucidated a well-integrated 2D NPLs-microbe interface facilitating rapid charge transfer. RNA sequencing (RNA-seq) and metabolomic studies disclosed a novel intracellular photoelectron energy transfer and conversion pathway for generating NADH and ATP, mediated by thiamine pyrophosphate (TPP), resulting in enhanced microbial energy metabolism (Fig. 1b). Our study reported the implementation of 2D nanomaterials in the cytosol as an artificial solar-energy system, and its application in achieving high yield of highly reduced chemicals in the cell factories. Results Dimensionality-dependent photoelectric conversion and cellular internalization In this study, we first investigated the influence of dimensionality on photoelectric conversion and cellular internalization rates performance using CdS nanocrystals (NCs) with different dimensions. We synthesized 0D QDs, 1D NRs, and 2D NPLs following previously reports 27, 28 . The morphology of these nanomaterials was characterized by transmission electron microscopy (TEM), confirming the dimensions: QDs with a diameter of 4.8 nm, NRs with dimensions of 20×5 nm, and 2D NPLs (4 ML) with dimensions of 40×40×2.2 nm (Fig. 2a). UV-visible (UV-vis) spectroscopy revealed maximum absorption peaks at 424 nm, 455 nm, and 424 nm for QDs, NRs, and NPLs, respectively (Supplementary Fig. 1). Photoelectric conversion efficiency was assessed via current-density-time (i-t) curves measured under blue light (470 nm) illumination. This method reflects the photocatalytic activity of the nanomaterials, a critical parameter in evaluating their performance. Photocurrent measurements under intermittent lighting conditions showed negligible current during “dark” periods, but significant photocurrent during “light” periods (Fig. 2b). The photocurrent data, as depicted in Fig. 2b, c, demonstrated that under blue light, NPLs exhibited a maximum photocurrent of 1.3 µA cm -2 , which was 3.79 times and 1.96 times higher than that of QDs and NRs, respectively (Fig. 2c). This enhancement can be attributed to the extensive 2D surface area and strong 1D quantum confinement in NPLs, which exceed 0D QDs and 1D NRs in promoting rapid charge transfer and effective charge separation 29, 30 . We next explored the uptake rate of nanomaterials by the bacterial cells, a critical step to achieve semiconductor biohybrid construction 22 . After introducing nanomaterials (1.5 mg L -1 of each) into a bacterial cell suspension and culturing in minimum medium (detail in the methods), cell growth was monitored by measuring the optical density at 600 nm (OD 600 ). The growth rates were comparable across all nanomaterial types and a control group without nanomaterials, indicating good biocompatibility of the 1.5 mg L -1 0D QDs, 1D NRs, and 2D NPLs with bacterial cells (Supplementary Fig. 2). Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the residual concentration of CdS nanomaterials in the minimum medium after 3 h of culture. Surprisingly, the residual concentrations of 1D NRs and 2D NPLs were notably lower at 20.74 ppb and 13.89 ppb, respectively, compared to 84.11 ppb for 0D QDs, indicating a higher uptake of NPLs by the bacteria (Fig. 2d). We attributed this high penetration rate to the unique flat structure of NPLs, which allowed better access to bacteria due to their ultra-thin longitudinal thickness and adaptability to various bacterial surface topographies under different mechanical conditions 31, 32 . Taking together, we speculated the superior photocurrent and better internalization of 2D NPLs may achieved a higher light-driven chemical production by photosensitizing bacteria. The atomic-layer thickness of 2D NPLs has been shown to control the optical and electronic properties, such as the carrier dynamics of two-dimensional NPLs 33 , thereby enhancing light absorption and facilitating effective charge separation 34 . Here, we investigated the effect of NPLs thickness (including 3, 4, and 5 ML) on photo-induced current efficiency (Fig. 3a, Supplementary Figs. 3 and 4). As shown in Fig. 2e, f, the photocurrent of 5 ML NPLs was 1.82 µA cm -2 , a 32.43% increase compared to 4 ML NPLs and a 1.16-fold increase compared to 3 ML NPLs, reflecting a decrease in two-dimensional bandgap with increasing thickness, which aids photogenerated electron mobility, particularly in 5 ML NPLs 35 . We also investigated the effect of NPLs thickness on internalization efficiency by measuring the residual concentration of Cd 2+ in the minimum medium (Fig. 2g). All thicknesses showed effective absorption, with the lowest residual concentration observed in 5 ML NPLs (8.14 ppb), albeit not significantly different from other thicknesses. Those results indicated the thicker 2D NPLs enhance photoelectrical efficiency while maintain the similar levels of bacterial cells internalization efficiency. Enhanced charge separation efficiency with core/crown heterojunction NPLs In addition to the electron transfer efficiency, as indicated by higher current density, the charge separation efficiency of a nanomaterial is another important factor influencing its photoelectrochemical performance. To improve charge separation efficiency and electron utilization performance, we modulated the energy level distribution by designing and constructing heterojunctions based on 5 ML NPLs (Fig. 3a). In comparison to other heterojunction nanomaterials, core-crown (CC) heterojunctions are capable of achieve efficient electron-hole separation by modulating the energy level distribution without necessitating changes to the thickness of the atomic layers or altering the optical absorption characteristics of the core NPLs. In this regard, zinc selenide (ZnSe) was chosen for its narrow band gap, and a CdS/ZnSe CC NPLs was constructed by epitaxial growth to create a type-II heterostructure. As shown in Fig. 3b, TEM images demonstrated the rectangular-shaped morphology of the NPLs with average lateral dimensions of 45×45×2.2 nm. In-situ energy-dispersive X-ray spectroscopy (EDS) mapping (Fig. 3c) provided further evidence for a core/crown composition in the CdS/ZnSe CC NPLs, with CdS (red and green) located in the central region and ZnSe (yellow and blue) at the outer edge. Raman spectroscopy measurements analyzed, the bonding composition of the heterojunction CC NPLs in detail, and identified a characteristic peak for the Cd-S bond at ~302 cm -1 (longitudinal optics, LO). X-ray diffraction (XRD) spectra demonstrated its crystalline characteristics of the zinc blende phase (Fig. 3e). The UV-vis and photoluminescence (PL) showed spectra shown a strong absorption peak at 440 nm and green emission peak at 510 nm (Fig. 3f). The energy differences between the valence (VB) and conduction (CB) bands reported in Fig. 3g, were based on UV photoelectron spectroscopy (UPS) measurements and calculations of the absorption and emission energies. The schematic representation in the insert of Fig. 3g described the band-edge alignment for CC NPLs. In the CC NPLs, charges were separated, with the electron wave function localized in the CdS core and the hole in the ZnSe crown. This type-II band alignment drives the photogenerated excitons at the ZnSe crown to migrate towards the core/crown interface, forming a charge-transfer (CT) exciton. The observed broader full width at half maximum (FWHM, 40 nm) was a common feature of type-II nanocrystals 36 . Subsequently, we measured the photocurrent of CC NPLs and the residual concentration of Cd 2+ in the minimum medium. As shown in Fig. 2e, f, the photocurrent of CC NPLs is 2.63 µA cm -2 , increased by 44.51% compared to 5 ML NPLs (1.82 µA cm -2 ), which illustrates that the core-crown heterojunctions (CdS/ZnSe CC NPLs) improve the charge separation efficiency. The residual Cd 2+ concentration of CC NPLs (9.71 ppb) after inoculated with bacteria was similar to 5 ML NPLs (8.14 ppb). Improved bioproduction of V. natriegens via CC heterojunction NPLs biohybrids V . natriegens , recognized as a next-generation host for biotechnology with exceptionally high growth (doubling time of less than 10 minutes) and substrate consumption rate 37 , has demonstrated the capability for extracellular electron transfer which could facilitate the transfer of photo-electrons from semiconductor to bacterial cells in biohybrid systems 38 . The bioproduction of BDO is dependent on the supply of reducing power, thus serving as an excellent platform for evaluating the energy efficiency of biohybrids. Thus, the pET-RABC plasmid 39 , which contains the BDO biosynthetic pathway, was introduced into V. natriegens , resulting in strain XG211. To test the hypothesis that enhanced internalization coupled with higher photocurrent of CC NPLs could improve bacteria photosensitization, we added the nanomaterials to the cell suspension (OD 600 ~ 2.0) of strain XG211 in the minimum medium with 4 g L -1 glucose, electron sacrificial agent (cystine) and mediator (flavin mononucleotide, FMN). After incubation for 1 h under blue light (470 nm, 5 mW cm - 2 ), the BDO production of strain XG211 were measured using gas chromatography (GC). We found that BDO production of strain XG211 with CC NPLs nanomaterials under illumination were all higher than their counterpart in absence of illumination or nanomaterials (Fig. 4b). Notably, BDO production in the strain with CC NPLs was superior to all other nanomaterials, achieving a final titer of 1.68 g L -1 (Fig. 4b), showing 1.28-fold increase compared with 5 ML NPLs without heterostructure (1.31 g L -1 ). Moreover, a series of control experiments were conducted to compare the light-driven BDO production with variations in nanomaterial dimensions. Initially, the BDO production of strain with 2D (4 ML) NPLs was 1.05 g L -1 , showing 35.4% and 36.3% increase when compared to 1D NRs and 0D QDs under illumination, respectively (Fig. 4a). Furthermore, 5 ML NPLs biohybrids achieve higher BDO production compared to 4 ML NPLs biohybrids, while the 3 ML NPLs biohybrids (0.94 g L -1 ) was similar to 4 ML NPLs biohybrids. As aforementioned mentioned, 2D nanomaterials offer better internalization coupled with enhanced photocurrent compared to 0D and 1D nanomaterials, resulting in higher BDO production. This trend is also the same regarding the layers of the nanomaterials. Taking together, our results demonstrated that all low-dimensional CdS nanomaterials were capable of photosensitizing V. natriegens , with 2D CC NPLs showing the highest efficiency in light-driven chemical production. After confirming the BDO production and the superior catalytic performance in 2D CC NPLs the biohybrids, we hypothesize a direct interaction at the nanomaterials-bacteria interface. To this end, we prepared cross-sectional slices of CC NPLs-XG211 biohybrids samples using microtome sections. High angle angular dark field-scanning transmission electron microscopy (HAADF-STEM) image and EDS mapping showed that internalized CC NPLs were composed of Cd, S, Zn and Se, with highly correlated locations (Fig. 4c and Supplementary Fig. 5). Those results clearly revealed the successful transportation of CC NPLs into the cytoplasm of V. natriegens . Previous studies have reported that intracellular photosensitizers enhanced the efficiency of photoelectron transfer and energy transduction by avoiding the energy loss during photoelectrons transmembrane transfer 40, 41 . We studied the charge-transfer kinetics at this internalized CC NPLs-XG211 biohybrids interface. The photo-excited electrons lifetime and photocurrent of both CC NPLs-XG211 biohybrids and pure CC NPLs were measured (Fig. 4d). The average photogenerated state lifetime of biohybrids was only 0.26 ± 0.01 ns, showing a 280-fold shorted compared with the CC NPLs alone (72.8 ± 5.2 ns) (Fig. 4e). Similarly, upon the addition of bacteria, the photocurrent of biohybrid systems decreased by about 69.96% compared to that of CC NPLs alone (Fig. 4f). These results indicated that the assembly of CC NPLs into bacteria results in rapid photo-induced charge transfer between CC NPLs and the bacterial cells. Photoelectron-induced regulation of energy metabolism in biohybrids The semiconductor harvested light for suppling reducing equivalent instead of sugar oxidation for microbial cells in the biohybrid systems, therefore increasing the carbon yield by reducing/eliminating carbon loss during chemical production (Fig. 5a). We firstly detail analyzed the biomass and glucose-to-BDO of strain XG211 with and without CC NPLs under illumination. The biomass of illuminated biohybrid systems was only slightly higher (~ 28.9% improvement) than all other conditions (Fig. 5b). However, the carbon yield of illuminated biohybrid systems reach 0.457 g g -1 , showing a 2.69-fold increase compared with XG211 with no nanomaterials, and a 2.01-fold increase compared to its counterpart under dark condition (Fig. 5c). Additionally, the production of acetoin (Fig. 1b), a direct precursor of BDO, was increased by 96.8% under light conditions (Supplementary Fig. 6). The mechanism of this enhanced production yield was further studied as followings. We initially measured the changes of intracellular energy pools including NAD(P) + , NAD(P)H, ADP and ATP. The NADH/NAD + ratio, represent the redox energy state of cells, in illuminated biohybrid systems surpassed all other conditions, with a ratio of 0.877 (Fig. 5d). This ratio indicated a 3.06-fold increase compared with its counterpart with no illumination and a 1.19-fold increase compared with bacterial system in absence of CC NPLs (Fig. 5d). While, the NADPH/NADP + ratio of illuminated biohybrid systems was 23% higher than that of dark condition and was 16% higher than that of bacterial system (Fig. 5d). The highest intracellular ATP concentration was also achieved in the illuminated biohybrid systems, increased 24% compared with the dark condition (Fig. 5e). Those results confirmed two-dimensional semiconductor biohybrid systems absorbed light to power the bacterial energy metabolism, thereby promoted the flux of BDO synthetic pathway which required quantity of reducing energy. To reveal the impacts of intracellular CC NPLs on bacterial metabolism during illumination, we performed metabolomics and transcriptomic analysis. We collected biohybrid samples with and without illumination for targeted metabolite quantification analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Central metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP), which were primary sources of reducing power and ATP, were analyzed. As shown in Fig. 5f, the intracellular concentration of key metabolites such as 6-phosphate glucose (G6P), fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3PG) and phosphoenolpyruvate (PEP) in the glycolysis, as well as Acetyl-CoA (AcCoA) and malate (MAL) in the TCA cycle, were increased under light compared to dark condition. Additionally, the concentration of 6-phosphogluconate (6-GPC) in the PPP pathway was also higher under light than dark condition (Fig. 5f). Those results suggested the nanomaterials induced photoelectrons likely improve the activity of central metabolism, which consistence with previous reports 19, 42 . To explored global regulation of bacterial cells by illuminated nanomaterials, we further performed RNA-seq to compare gene expression and transcription activation at genome-wide level. Comparing biohybrid with versus without illumination, we identified 28 genes with significantly upregulation and 28 downregulation (log 2 (fold change (FC)) > 1.0 or < -1.0 and P < 0.01) (Fig. 5g, h). Gene ontology (GO) term analysis of the upregulated genes highlighted a common feature: “sulfide metabolism” or “flavin binding” (Supplementary Fig. 7), which likely related to electron acceptors or mediators participating in bacterial electron transfer processes. As shown in Fig. 5g, h, the upregulated genes including those encoding proteins involved in cellular energy metabolism with illumination, such as the oxidative respiratory chain (Complex Ⅰ [NADH-quinone reductase], Complex Ⅱ [fumarate reductase], Complex Ⅳ [cytochrome ubiquinol oxidase]), FAD [FMN transferase] and RNF [RNF complex, electron transport complex], as well as the ATPase [ATP synthase]. Remarkably, 10 out of 28 upregulated genes were involved in thiamine biosynthesis, for production of TPP (the active form of thiamine), a key cofactor for enzymes involved in central metabolic pathway such as transketolase and dehydrogenase, which participate in PPP, TCA cycle and the link between glycolysis and TCA cycle 43-45 . Those enzymes are vital for the production of NADH, NADPH and ATP. The above up-regulation genes were further confirmed by the real-time fluorescence quantitative PCR (qRT-PCR) results, which consistence with the RNA-seq results (Supplementary Fig. 8). Taking together, those results suggesting the illuminated biohybrid systems not only provide extra-energy from light but also promote both carbon metabolism and energy metabolism of bacteria, notably increasing carbon yield by reducing or eliminating carbon loss during chemical production. Verification and application of photoelectron-induced regulation in biohybrids In our biohybrid systems, semiconductors capture light to supply reducing equivalents for cellular metabolism, significantly enhancing the thiamine/TPP synthetic pathway. This pathway typically boosts the native energy machinery (central metabolism) to generate more reducing power. Observations of these effects led us to hypothesize that converting photoelectrons into reducing power might require the mediation of TPP. To explore this hypothesis, we analyzed the rate of NADH generation from CC NPLs with and without TPP under in vitro conditions (Fig. 6a, b). In a typical experiment, 1.5 mg mL -1 CC NPLs, 1 mM NAD + with or without TPP was inoculated under 470 nm light (5 mW cm -2 ). NADH regeneration was monitored by the absorption at 340 nm (Abs 340 nm) and the final NADH product was confirmed by nuclear magnetic resonance (NMR, Fig. 6b). Adding TPP resulted in a 4.45-fold increase in NADH production under light conditions compared to controls without TPP, whereas NADH production remained unchanged under dark conditions (Fig. 6c). These findings were corroborated by NMR, affirming that the increase in Abs 340 nm was indeed attributable to heightened NADH production (Fig. 6b). Additionally, the presence of thiamine (vitamin B1), commonly used as a substrate in cultures for TPP biosynthesis, led to a 9.45-fold increase in NADH production under light conditions, with no improvement noted under dark conditions (Supplementary Fig. 9). Based on these results and corresponding in vitro experiments, both TPP and thiamine not only enhance reducing power production from photoelectrons but also boost the native central metabolism. Recent studies also suggest that TPP/thiamine promotes NADH production and electron transfer in two bacterial species co-cultures 46 . We speculated the photoelectron transfer and solar-to-chemical efficiency in biohybrid systems might enhanced by the addition of TPP/thiamine. To investigate further, we cultured biohybrid systems with varying concentrations (0, 0.3, 0.6, 1, 3, 6 mM) of TPP or thiamine, analyzing the ratio of BDO production and carbon yield of illuminated biohybrids compared to control bacteria (Fig. 6d-6f), and also compared biohybrid performance under light versus dark conditions (Fig. 6g-6i). The ratio of BDO production and carbon yield of illuminated biohybrids increased by 11.73% and 8.57% respectively with 0.6 mM TPP compared to the conditions without TPP, showing a decrease when TPP concentrations exceeded 1 mM (Fig. 6e, f). Similarly, the ratio of BDO production and carbon yield of the biohybrid systems under light increased by 22.75% and 24.02% with 0.6 mM TPP compared to conditions without TPP, decreasing with higher TPP concentrations (Fig. 6h, i). However, the growth of biomass was increased 38.09% and 21.29% with 6 mM TPP under light and dark compared to the control without TPP, respectively (Supplementary Fig. 10). Similar results were observed with thiamine, where both BDO production and carbon yield increased with 0.6 mM thiamine (Supplementary Fig. 11). These results indicate that TPP/thiamine indeed promotes solar-to-chemical efficiency, but the concentration is critical for optimizing carbon partitioning between desired chemicals and biomass growth. Verification and application of photoelectron-induced regulation in biohybrids In our biohybrid systems, semiconductors capture light to supply reducing equivalents for cellular metabolism, significantly enhancing the thiamine/TPP synthetic pathway. This pathway typically boosts the native energy machinery (central metabolism) to generate more reducing power. Observations of these effects led us to hypothesize that converting photoelectrons into reducing power might require the mediation of TPP. To explore this hypothesis, we analyzed the rate of NADH generation from CC NPLs with and without TPP under in vitro conditions (Fig. 6a, b). In a typical experiment, 1.5 mg mL -1 CC NPLs, 1 mM NAD + with or without TPP was inoculated under 470 nm light (5 mW cm -2 ). NADH regeneration was monitored by the absorption at 340 nm (Abs 340 nm) and the final NADH product was confirmed by nuclear magnetic resonance (NMR, Fig. 6b). Adding TPP resulted in a 4.45-fold increase in NADH production under light conditions compared to controls without TPP, whereas NADH production remained unchanged under dark conditions (Fig. 6c). These findings were corroborated by NMR, affirming that the increase in Abs 340 nm was indeed attributable to heightened NADH production (Fig. 6b). Additionally, the presence of thiamine (vitamin B1), commonly used as a substrate in cultures for TPP biosynthesis, led to a 9.45-fold increase in NADH production under light conditions, with no improvement noted under dark conditions (Supplementary Fig. 9). Based on these results and corresponding in vitro experiments, both TPP and thiamine not only enhance reducing power production from photoelectrons but also boost the native central metabolism. Recent studies also suggest that TPP/thiamine promotes NADH production and electron transfer in two bacterial species co-cultures 46 . We speculated the photoelectron transfer and solar-to-chemical efficiency in biohybrid systems might enhanced by the addition of TPP/thiamine. To investigate further, we cultured biohybrid systems with varying concentrations (0, 0.3, 0.6, 1, 3, 6 mM) of TPP or thiamine, analyzing the ratio of BDO production and carbon yield of illuminated biohybrids compared to control bacteria (Fig. 6d-6f), and also compared biohybrid performance under light versus dark conditions (Fig. 6g-6i). The ratio of BDO production and carbon yield of illuminated biohybrids increased by 11.73% and 8.57% respectively with 0.6 mM TPP compared to the conditions without TPP, showing a decrease when TPP concentrations exceeded 1 mM (Fig. 6e, f). Similarly, the ratio of BDO production and carbon yield of the biohybrid systems under light increased by 22.75% and 24.02% with 0.6 mM TPP compared to conditions without TPP, decreasing with higher TPP concentrations (Fig. 6h, i). However, the growth of biomass was increased 38.09% and 21.29% with 6 mM TPP under light and dark compared to the control without TPP, respectively (Supplementary Fig. 10). Similar results were observed with thiamine, where both BDO production and carbon yield increased with 0.6 mM thiamine (Supplementary Fig. 11). These results indicate that TPP/thiamine indeed promotes solar-to-chemical efficiency, but the concentration is critical for optimizing carbon partitioning between desired chemicals and biomass growth. Discussion In this work, we report a 2D nanomaterials-biohybrid systems that achieve higher carbon yield and solar-to-chemical efficiency. Through the systematic exploration, our findings reveal that 2D CC NPLs nanomaterials exhibit superior photoelectrochemical efficiency and internalization rates compared to 0D and 1D nanomaterials, consequently yielding the highest production yield of chemical. The intracellular 2D nanomaterials offer a closed nanomaterial-microbe interaction, generate photoelectrons in the cytosol, thereby bypassing energy-consuming cross membrane electron transfer and facilitating faster electron conversion. Unlike previous studies focused on extracellular or periplasmic electron transfer pathways (Supplementary Table 2), our research unveils a novel intracellular photoelectron energy conversion pathway. Transcriptomic analysis has shown an upregulation of the TPP biosynthetic pathway and the oxidative respiratory chain. Moreover, in vitro assays have demonstrated TPP promotes NADH generation, suggesting TPP mediates the transfer and conversion of photoelectron energy from the nanomaterials to NADH and ATP. Those findings will guide the rational engineering of semiconductor biohybrid systems to approach the maximum solar-to-chemical efficiency during bioproduction process. TPP is a key cofactor for enzymes involved in central metabolic pathways such as glycolysis, pentose-phosphate pathway and the citric acid cycle, all vital for the bioproduction of ATP and NAD(P)H 47 , thus it’s essential to all life. Here, we discovered that TPP also plays a role in artificial photoelectron energy conversion. Future detailed studies should explore the mechanisms of TPP-mediated photoelectron transfer, such as the number of electrons each TPP molecule can carry, and how native catabolic processes are cross-regulated with photoelectrons to provide energy and metabolic precursors for chemical production. Our preliminary data indicate that varying TPP concentrations differently regulate BDO production and biomass growth in the light-driven semiconductor biohybrid: lower TPP concentrations enhance BDO production, whereas higher concentrations promote biomass growth (Fig. 6 e, h and Supplementary Fig. 10). Those results suggest TPP plays a key role in partitioning carbon flux towards biomass and desired chemicals. Moving forward, we need to understand the regulatory mechanisms of TPP on carbon and energy metabolism to achieve the maximum chemical productivity and carbon utilization efficiency, ultimately leading a higher solar-to-chemical conversion efficiency. Methods Synthesis and p urification of various nanocrystals CdS QDs. CdS QDs were prepared according to the reported procedure 48, 49 . Cd-precursor: a mixture of 0.256 g CdO, 6.3 mL oleic acid and 13.7 mL ODE was heated in a round-bottomed flask at 250 °C under N 2 flow until the mixture became clear. S-precursor: a mixture of 64 mg of S and 20 mL of ODE was heated in a round-bottomed flask at 180 °C until the mixture became clear. The mixture of 4 mL of Cd-precursor, 6 mL of ODE was bubbled with N 2 , then heated to 265 °C from room temperature. 2 mL of S-precursor was injected into the mixture and the temperature was reduced to 220 °C and held at this temperature for 100 seconds. The reaction was then carried out at 220 °C for 1 minute with the addition of 1 mL of S-precursor and 1 mL of Cd-precursor. By repeating this process, the QDs can be brought to the desired size (2 times for CdS QDs with diameter of 4.8 nm) and then the mixture is cooled to room temperature to stop the reaction. After the reaction, the liquid mixture was transferred to a centrifuge tube and acetone (three times the volume of the reaction mixture) was added to precipitate the QDs. The mixture was shaken well and centrifuged at 5,000 rpm for 10 minutes. The supernatant was discarded and the precipitate redispersed in hexane. A threefold excess of methanol was added, the mixture was shaken well and centrifuged at 5,000 rpm for 10 minutes. Repeat the above purification and store in hexane. CdS rods (50.0 ± 3.2 × 5.0 ± 0.3 nm). CdS rods samples were synthesized using a previously reported procedure 28, 50 . For the reaction, a 25 mL 3-necked flask was filled with 210 mg of CdO and 2.75 g of TOPO. The 0.8 g ODPA and 0.22 g TDPA were used. The mixture was evacuated at 120 °C for 30 minutes and then heated to 320 °C under N 2 for 15 minutes to allow complexation of cadmium with phosphonic acid. To remove water formed during complexation, the reaction mixtures were cooled to 120 °C and evacuated again for 1 h. While reheating to 320 °C, 2 g of TOP was injected into each flask. 1.3 g TOP-S (TOP and elemental S in a 1:1 molar ratio) was then injected and the nanocrystals were grown at 315 °C for 85 minutes. After cooling, the nanorods were washed several times by adding equal amounts of nonanoic acid and isopropanol, followed by centrifugation to precipitate the CdS nanorods. The supernatant was removed, and the precipitated nanorods were redispersed in fresh toluene. 3 ML NPLs. The CdS NPLs were synthesized according to the reported procedure 51 . The S-ODE solution was prepared by dissolution of 0.0320 g of sulfur (1.0 mmol) in 20 g of ODE by gentle sonication and stored in a closed vial. Cadmium acetate dihydrate (0.1066 g, 0.4 mmol), S-ODE (2.0032 g, 0.1 mmol S), myristic acid (0.0914 g, 0.4 mmol) and 6.0 g ODE were bubbled with N 2 for 10 minutes, then heated from room temperature to 180 °C in 10 minutes and kept under N 2 flow for a further 30 minutes. 4 ML NPLs. Cadmium acetate dihydrate (0.1066 g, 0.4 mmol), S-ODE (2.0032 g, 0.1 mmol S), oleic acid (0.113 g, 0.4 mmol) and 6.0 g ODE were bubbled with N 2 for 10 minutes. After switching to N 2 protection, the solution was heated from room temperature to 260 °C in 15 minutes and held at 260 °C for 1 minute. 5 ML NPLs. CdO and stearic acid (3 times the molar ratio of CdO) were first bubbled with N 2 for 30 minutes, then heated to 160 °C and kept at this temperature until the color of the mixture became almost transparent after about 2 h of reaction. During the cooling process, the solution was added to a large beaker of hot acetone, kept at 55~60 °C, and stirred vigorously for about 10~15 minutes. The white precipitates were separated from the supernatant by timely vacuum filtration. The purification procedure was repeated four more times before the precipitates (Cd(St) 2 ) were placed in a vacuum oven for overnight pumping treatments at room temperature. Cd(St) 2 (0.1356 g, 0.2 mmol), S-ODE (2.0032 g, 0.1 mmol S) and 6.0 g ODE were bubbled with N 2 for 10 minutes, then heated from room temperature to 190 °C within 8 minutes and then held at the specified temperature under N 2 flow for 8 minutes. After cooling to room temperature, 0.1066 g of cadmium acetate dihydrate (0.4 mmol) was added, the solution was bubbled with N 2 for 10 minutes and then heated from room temperature to 250 °C in 15 minutes and held at 250 °C for a further 1 minute. Purification of CdS NPLs of various thicknesses. The CdS NPLs were mixed with chloroform and oleylamine (10% volume ratio of oleylamine) to form a clear solution. A mixture of acetone and methanol (30% volume ratio of methanol and 5 times volume of chloroform/oleylamine) was then added, and after sonication for 1 minute, the solution was centrifuged at 11,000 rpm for 5 minutes. The supernatant was discarded and the precipitates were collected for a further three rounds of the above purification and redispersed in fresh toluene. CC NPLs. CC NPLs were synthesized following a method from the literature with slight modifications. The TOP-Se (1 M) solution was prepared by dissolving selenium in TOP with vigorous stirring and stored in a sealed vial under N 2 . 5 ML NPLs dissolved in 5 mL ODE, 238 μL (0.75 mmol) oleic acid and 109.7 mg (0.5 mmol) zinc acetate were placed in a 25 mL four-necked round bottom flask and degassed under vacuum for 60 minutes at room temperature. The reaction mixture was heated to 255 °C under N 2 flow and after reaching this temperature, 50 μL of 1 M TOP-Se (0.05 mmol Se) in 1 mL ODE was rapidly injected into the reaction mixture. Allow to react for 20 minutes, then cool to room temperature in a water bath. The product is dispersed in hexane and then purified with ethanol by two rounds of centrifugation and finally redispersed in toluene. Phase transfer of these CdS nanocrystals to water The phase transfer of the sample is carried out according to the reported ligand exchange method with slight modifications 52 . In a typical procedure, 1 mL of nanocrystals dispersion in toluene (around 5 mg mL -1 ) was combined with 0.2 mL DMF solution of NOBF 4 (10 mg mL -1 ) at room temperature. The resulting mixture was gently shaken until the precipitation of the nanocrystals could be observed, which was usually within 5 minutes. After centrifugation to remove the supernatant, the precipitated nanocrystals can be redispersed in the hydrophilic media DMF. To remove the excess ligands, DMF and toluene (1:1 v/v) were added to flocculate the nanocrystals dispersion. After centrifugation, the nanocrystals can be redispersed by adding the polar solvents mentioned above to form a stable colloidal dispersion. The hydrophilic nanocrystals obtained by NOBF 4 treatment can be further functionalized by other capping molecules through secondary ligand exchange reactions, achieving a complete phase transfer of nanocrystals between hydrophobic and hydrophilic. The NOBF 4 -treated nanocrystals were then redispersed in 1 mL of an aqueous solution of L-cysteine (1 mg mL -1 , adjusted to pH > 10 with NaOH). Subsequent shaking (overnight) allowed the L- cysteine to successfully bind to the surface of the nanocrystals. The nanocrystals were precipitated by the addition of ethanol. The precipitated nanocrystals were redispersed in water to form a stable colloidal dispersion after centrifugation to remove the supernatant. Characterization of nanocrystals All measurements were carried out at room temperature. The absorption spectra of the nanocrystals were recorded using a Carry 5000 (Agilent). The PL, PLE and lifetime were measured on a HORIBA FL-3 3D fluorescence spectrometer. Raman scattering information from the sample was collected using a laser confocal Raman spectrometer (Renishaw InviA-Reflex). A JEOL JEM-2800 transmission electron microscope (TEM) was employed for TEM, scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDS) mapping investigations. A Bruker D8 Advance diffractometer equipped with Ni-filtered Cu Kα radiation was used to collect the powder X-ray diffraction (XRD) data. For powder X-Ray diffraction (PXRD), samples are prepared by evaporating the solvent directly from the concentrated dispersion onto a single crystal silicon sample holder. Culture medium and condition (1) Rich culture medium LBv2 (per 1 L) : 25 g LB powder, 11.9 g NaCl, 0.313 g KCl, 2.2 g MgCl 2 , and all solidified media for bacterial growth contain 1.5% [w/v] agar. (2) VN minimum medium (per 1 L) (pH=7.5) : 5 g (NH 4 ) 2 SO 4 , 15 g NaCl, 1 g KH 2 PO 4 , 1 g K 2 HPO 4 , 0.25 g MgSO 4 , 0.036 g CaCl 2 ·2H 2 O, 16.4 mg FeSO 4 ·7H 2 O, 4 g glucose, 10 mg MnSO 4 ·H 2 O, 0.3 mg CuSO 4 ·5H 2 O, 1 mg ZnSO 4 ·7H 2 O, 0.02 mg NiCl 2 ·6H 2 O. V. natriegens cells were cultured overnight in LBv2 media containing appropriate antibiotics (kanamycin 200 μg mL -1 and chloramphenicol 30 μg mL -1 ). For the main culture, 100 mL of LBv2 medium was inoculated with 1 mL of the above preculture and cultured for the desired time (3 h) at 37 °C with agitation at 220 rpm. Construction of the BDO producing strain The electrocompetent cells of V. natriegens cells were prepared as described previously 42 . The pET-RABC plasmid was introduced via electroporation. Gently mix 200 ng of plasmid pET-RABC with 100 µL of electrocompetent cells in a chilled 1.5 mL microcentrifuge tube. Transfer the cell-DNA suspension to a cooled electroporation cuvette with a 0.2 cm gap. Electroporate cells at 1.7 kV and immediately recover them in 500 μL of recovery medium (BHI supplemented with v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl 2 , and 680 mM sucrose)). Incubate the cells at 37 °C for 1 h, then plate them on agar plates containing appropriate antibiotics. Incubate the plates at 37 °C overnight for colony growth. Construction of biohybrid systems and BDO production The strain XG211 was inoculated in VN medium supplemented with 1.5 mg L -1 CdS, and the OD 600 was adjusted to 0.1. The aerobic cultivation was conducted in an incubator with the temperature set to 37 °C. The cells were collected after 3 h, washed once with VN medium and adjusted to OD 600 of ~2.0. Add 1 mM cysteine as electron sacrificial agent and 5 µM flavin mononucleotide (FMN) as mediator. React in 5 mW cm - 2 blue light (470 nm) for 1 h at 37 °C with agitation at 200 rpm, and take the supernatant for GC test, collect bacteria for metabolite test. Photoelectrochemical analysis The UV-vis (Carry 5000 (Agilent)) was used to measure the direct bandgap. The dispersed nanoparticles or semiconductor biohybrids were loaded on carbon paper, respectively, to form a uniform 1 × 1 cm 2 film by drop casting and vacuum drying. The photoelectrochemical measurements were performed by a standard three-electrode configuration in electrolyte (PBS). Ag/AgCl (3 M NaCl) and Pt wire served as the reference and counter electrode, respectively. A 470 nm blue light panel was employed as the laser, with the light intensity calibrated to 5 mW cm - 2 . Current densities were recorded under 0.5 V bias vs Ag/AgCl. Measuring the remaining Cd 2+ content The XG211 strain was cultured in a rich culture medium at 37 °C for approximately 3 h. Subsequently, 1% of the bacterial culture was transferred into fresh VN medium and grown at 37 °C for an additional 3 h. The initial optical density at OD 600 being approximately 0.1. During this culture period, three different types of nanomaterials, namely 0D QDs, 1D NRs, and 2D NPLs, were separately added to the VN medium at a concentration of 1.5 mg L -1 . The supernatants were collected to measure the removal efficiency using ICP-MS (Inductively Coupled Plasma Mass Spectrometry) with a NexION 1000 instrument from PerkinElmer. Quantifying intracellular metabolites in biohybrids To quantify intracellular metabolites, the extraction method was adapted from Gao et al 42 . Specifically, cells were collected by centrifugation at 13,000 rpm for 10 minutes at 4 °C and immediately resuspended in 1 mL of methanol/water (80:20, vol/vol) pre-cooled at –80 °C. The samples were centrifuged at 13,000 rpm for 10 minutes at 4 °C. The supernatants were collected for quantification of metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS, SCIEX Triple Quad™ 5000+ QTRAP® Ready, AB SCIEX, USA). Quantifying BDO and glucose To determine the BDO concentration, the bacterial culture was centrifuged at 12,000 rpm for 10 minutes, and the same volume of supernatant and isometric ethyl acetate was mixed for sonication for 20 minutes and centrifuged for 10 minutes. Supernatant was filtered using nylon membrane (0.22 μm) and then determined using gas chromatography (GC). The GC system (Agilent 8890, Agilent Technologies Inc., USA) is equipped with a DB-WAX capillary column (30 m × 0.53 mm × 1 μm) and a flame ionization detector. Hydrogen gas was used as the carrier gas. The injector and detector temperature were maintained at 250 °C, and the oven temperature was 80 °C. The injection volume was 1 μL. To obtain the calibration curves for quantifying BDO and acetoin, a series of standards were quantified by GC system after the same pretreatment. Glucose content was determined using glucose assay reagent (Beyotime, China). Extracellular NADH regeneration assay In 1 mL of VN medium containing 1 mM NAD + , 1.5 mg mL -1 of CC NPLs, 1 mM cysteine as a sacrificial agent, and 5 μM FMN as a mediator were added. After a 2 h reaction, the absorbance at 340 nm in the test solution was measured to calculate the amount of NADH generated. The impact of adding 0.06 mM thiamine or 1 mM TPP to the above solution on NADH production was observed. Nuclear magnetic resonance (NMR) was employed for the quantification of NADH content in the test solution. The test solution comprised 450 μL of sample solution and 50 μL of D 2 O. NMR data were acquired using either the Bruker AVANCE NEO 400 or AVANCE III HD50 spectrometer. The collected data were processed and integrated using Bruker TopSpin software. Determination of quantum efficiency The calculation methods of quantum yield (QE) were mostly applicable to monochromatic light or single wavelength 22 , such as where N a is Avogadro’s constant (6.02×10 23 ) , C a represents the difference in the molar amount of BDO production between biohybrid systems and non-hybrid systems. V=total suspension vol (1 mL). Φ ph is the measured average photon flux, Φ ph cm -2 s -1 = 1.18×10 14 cm -2 s -1 . t=reaction time (3600 s). A=area of illumination (1.4 cm 2 ). Acetoin+NADH+H + → BDO+NAD + NAD + +2e - +H + →NADH Acetoin+2e - +H + →BDO RNA sequencing ( RNA seq ) CC NPLs-XG211 biohybrids were cultured in VN medium under both light and dark conditions for 1 h. Subsequently, the harvested cells were collected by centrifugation (4,000 rpm, 4 °C, 20 minutes) and flash-frozen in liquid nitrogen. RNA-seq experiments were conducted by Sangon Biotech (Shanghai) Co., Ltd. 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Brown, K.A., Wilker, M.B., Boehm, M., Dukovic, G., King, P.W. Characterization of photochemical processes for H 2 production by CdS nanorod-[FeFe] hydrogenase complexes. J. Am. Chem. Soc. 134, 5627–5636 (2012). Zhang, Y., et al. Engineering of exciton spatial distribution in CdS nanoplatelets. Nano Lett. 21, 5201–5208 (2021). Dong, A., et al. A generalized ligand-exchange strategy enabling sequential surface functionalization of colloidal nanocrystals. J. Am. Chem. Soc. 133, 998–1006 (2011). Declarations Acknowledgements We extend our gratitude to Prof. Ping Xu’s group for their generous provision of the pET-RABC vector containing BDO biosynthetic genes. We acknowledge the Shenzhen Infrastructure for Synthetic Biology for instrument support and technical assistance. This work was supported by the National Natural Science Foundation of China (Grant No. 32230060, 32171426, 22171132), the National Key R&D Program of China (Grant No. 2021YFA0910800, 2021YFA0909900, 2022YFC3401802), Shenzhen Science and Technology Program (Grant No. JCYJ20220818101804010, RCYX20221008092901004, ZDSYS20220606100606013, KJZD20230923114419039 and JCYJ20220531100006011) and the Natural Science Foundation of Guangdong Province, the Science and Technology Program in Jiangsu Province (BK20232041), the Program for Innovative Talents and Entrepreneurs in Jiangsu (020513006012 and 020513006014) and the Zijin Scholars Foundation (0205181022). Author Contribution The project was conceptualized by Y. W. and X.G., and was supervised C. Y., X. G., Y. W.. X. K. prepared the materials and performed the characterization. M. G. performed the experiments of the biohybrid construction and characterization. W. C. performed the metabolomics. W. Y. and X. Y. performed the experiment of CRISPRi. X. W. and S. P. performed the experiment of photocurrent measurement. M. G., X. K., Y. W., and X. G. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript. Declaration of Interests All authors declare no competing interests. Data availability All data presented in this manuscript are available in the paper and its Supplementary Information. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterials20240516.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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China","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xiang","suffix":""},{"id":303293356,"identity":"f10d27c7-f46a-4b18-a142-d66d2b97a1a1","order_by":8,"name":"Fangfang Duan","email":"","orcid":"","institution":"Department of Pharmacology, School of Medicine, Shenzhen Campus of Sun Yat-Sen University, Shenzhen 518107, P. R. China","correspondingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Duan","suffix":""},{"id":303293357,"identity":"4ae38700-a331-4d2e-b52d-2e7f85e2dcc0","order_by":9,"name":"Chen Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYPACGwbGBhDNRryWNAbGNhK1HIaqJkaLwfGzh18w1Jy3Z57fY8DwoewwA//sBgJazuSlWTAcu53Y2MZjwDjj3GEGiTsHCGg5kGNmwNhwO4ERqIWZt+0wg4FEAgEt59+AtJyzB2v5S5SWGznGDxgbDjCCHMbMSIwWyRtvzBgSjiUD/ZJWcLDnXDqPxA0CWvjO5xh/+FBjZ2/YfHjjgx9l1nL8MwhoUTjAwAZ2iWEDA8MBIM2DXz0QyDcwMH8AMwgqHQWjYBSMghELAIgAQqATalP5AAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China","correspondingAuthor":true,"prefix":"","firstName":"Chen","middleName":"","lastName":"Yang","suffix":""},{"id":303293358,"identity":"d2df981a-b93f-4a63-bda8-1c6cc967bb7b","order_by":10,"name":"Yuanyuan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYPACGwbGBiDFQ4KWNNK1HIZQRGkxOH728GvetvN5zDMSGB+8bWOQNyeo5UxemjVv2+1ixhkJzIZz2xgMdzYQ0GJ2IMfMGKglsXFGAps0bxtDgsEBQlrOvwFpOQfSwv6bOC03cowf87YdANvCTJQW+xtvzBjnnEtObOx52Cw555yE4QZCWiT7c4w/vCmzS9zYnnwQyLCRJ2gLELBJ8bIxMBg2gCNTgrB6IGD++OMPA4M8UWpHwSgYBaNgRAIAN3lDHZiyHQsAAAAASUVORK5CYII=","orcid":"","institution":"State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China","correspondingAuthor":true,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Wang","suffix":""},{"id":303293347,"identity":"2f7fca1f-df7b-420e-94c9-a8920c0d5d5d","order_by":11,"name":"Xiang Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie2NsQrCMBRFnwh1KXRNp/xCiiBO7ehvJAScxLmgYEToaD9GcI4E2qXimsFBEDo5CII4iZHujW6CORDCg3s4AA7Hr0LNC6AjvlRCYRT5VYm85x8peKQKdJof481BLW9XiDH09u1mVI05oUXNt5qtkAQeCX9K2xUxiU7UU3ygO8IoXQrIJ+1KfiGSPhXv57vVQ8LCrmBkKixTMQGWmYqyKwTVnLC1okizbFiRMsr8iaWS8yJ83FUS5OVZp+kMB73KUpHNz0RzAnit+3elmUJiGzocDscf8wI8SEf//Ofx5wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0699-0351","institution":"Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academic of Science, Shenzhen 518000, P. R. China","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-05-16 14:16:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4431666/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4431666/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56885312,"identity":"b0ee48a1-3d47-4628-807f-5c02bea6a233","added_by":"auto","created_at":"2024-05-21 18:29:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":283733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of TPP-mediated solar-driven production of BDO in semiconductor-engineering \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. natriegens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biohybrid systems with varying CdS dimensions. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eTrends in BDO production of residual nanomaterials, photocurrent of different dimensions and biohybrid systems in CdS nanomaterials of varying dimensions of biohybrid systems. Residual nanomaterials: the relative residual concentration of each nanomaterial in minimum medium after 3 h of co-culture with bacteria. Photocurrent, the relative photocurrent of each nanomaterial. BDO production, the relative production of BDO under light by engineered bacteria \u003cem\u003eV. natriegens\u003c/em\u003e in the presence of 0D QDs, 1D NRs, 2D NPLs (3 ML, 4 ML, 5 ML, and CC NPLs). \u0026nbsp;3 ML, 3-monolayers nanoplatelets; 4 ML, 4-monolayers nanoplatelets; 5 ML, 5-monolayers nanoplatelets; CC NPLs, core-crown of 5 ML nanoplatelets. (\u003cstrong\u003eb\u003c/strong\u003e) Left: pseudo-colored transmission electron microscopy (TEM) image of the CC NPLs structured biohybrids. Middle: schematic illustration of TPP-mediated electron transfer from CC NPLs to bacteria for BDO synthesis. Right: schematic diagram of the electron transfer process within the biohybrid systems. Proposed three possible electron transfer pathways in biohybrid: ①: Synthesis of NADH in glycolysis. ②: Synthesis of ATP in the ETC. ETC, electron transport chain in oxidative phosphorylation. ③: Regeneration of NADH during the synthesis of BDO from acetoin. \u003cem\u003ehv\u003c/em\u003e, incident photon; e\u003csup\u003e-\u003c/sup\u003e, reducing equivalent; Pyr: pyruvate; ADP/ATP, adenosine diphosphate/triphosphate; BDO, 2, 3-butanediol; TPP, thiamine pyrophosphate; RNF, electron transport complex; Complex I, NADH-quinone oxidoreductase of ETC; Complex II, succinate dehydrogenase of ETC; Complex IV, cytochrome c oxidase of ETC; ATPase, ATP synthase.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/afda8563ad734c785ceac3d6.png"},{"id":56885070,"identity":"c6084014-900e-4e1f-bf18-e3fdc6b2f669","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":571657,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and heterostructure performance characterization of low-dimensional semiconductors. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) TEM images of 0D, 1D and 2D (4 ML) semiconductors. (\u003cstrong\u003eb, c\u003c/strong\u003e) Photocurrent response in 0D, 1D and 2D (4 ML) semiconductors (470 nm, 5 mW cm\u003csup\u003e-2\u003c/sup\u003e). Materials concentration: 1.5 mg L\u003csup\u003e-1\u003c/sup\u003e. Error bar: standard deviation, n = 4. (\u003cstrong\u003ed\u003c/strong\u003e) After co-incubation of 0D, 1D, and 2D (4 ML) semiconductors with bacteria for the 3 h, the residual amount of Cd\u003csup\u003e2+\u003c/sup\u003e in the minimum medium. Error bar: standard deviation, n = 4. (\u003cstrong\u003ee, f\u003c/strong\u003e) Photocurrent response in 3 ML, 4 ML, 5 ML NPLs and CC NPLs (470 nm, 5 mW cm\u003csup\u003e-2\u003c/sup\u003e). Materials concentration: 1.5 mg L\u003csup\u003e-1\u003c/sup\u003e. Error bar: standard deviation, n = 4. (\u003cstrong\u003eg\u003c/strong\u003e) After co-incubation of 3 ML, 4 ML, 5 ML and CC NPLs semiconductors with XG211 for the same duration, the residual amount of Cd\u003csup\u003e2+\u003c/sup\u003e in the minimum medium. Error bar: standard deviation, n = 4. P values are determined by a two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/2e5a7799cbdec335c60d7be5.png"},{"id":56885077,"identity":"cf2b7bbd-de61-4c34-8995-40d0aa02df0f","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":686473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of CC NPLs semiconductors. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) The rational design of semiconductors based on their varied dimensions and charge separation. (\u003cstrong\u003eb, c\u003c/strong\u003e) High angle angular dark field-scanning transmission electron microscopy (HAADF-STEM) image and EDS mapping of CC NPLs. (\u003cstrong\u003ed\u003c/strong\u003e) Raman spectrum of 2D NPLs (bottom trace) and CC NPLs (upper trace). (\u003cstrong\u003ee\u003c/strong\u003e) XRD pattern of CC NPLs. (\u003cstrong\u003ef\u003c/strong\u003e) UV-vis absorbance and PL emission spectra of CC NPLs (with sample photograph insert). (\u003cstrong\u003eg\u003c/strong\u003e) The Tauc plot of CC NPLs. The intercept was employed to determine the direct band gap. Inset: band structure information for CC NPLs, where CB represents the conduction band and VB denotes the valence band. α is the absorption coefficient, h is Planck’s constant (6.626 × 10\u003csup\u003e−34\u003c/sup\u003e J·s), and v is the frequency. P values are determined by a two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/aeff68fc20aa0db30355ff2b.png"},{"id":56885072,"identity":"30a94d9a-2537-4188-b966-7aa3e858b68d","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe photocatalytic performance and characterization of the CC NPLs-XG211 biohybrids. \u003c/strong\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) BDO production of 0D, 1D, 2D, 3 ML, 5 ML NPLs and CC NPLs semiconductors-XG211 biohybrid systems under light (470 nm, 5 mW cm\u003csup\u003e-2\u003c/sup\u003e) and dark. Error bar: standard deviation, n = 4. (\u003cstrong\u003ec\u003c/strong\u003e) HAADF-STEM image and EDS mapping of the CC NPLs-XG211 biohybrid systems. (\u003cstrong\u003ed\u003c/strong\u003e) Schematic illustration of the CC NPLs-XG211 biohybrids interface (not drawn to scale). ET: electron transfer. PL: photoluminescence. (\u003cstrong\u003ee\u003c/strong\u003e) Time profiles of normalized transient absorption and the lifetime of biohybrids photoexcited states as determined by time-correlated single-photon counting. (\u003cstrong\u003ef\u003c/strong\u003e) Photoelectrochemical measurements of BDO cells, CC NPLs, and CC NPLs-XG211 biohybrid systems. P values are determined by a two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/ba48c2d71c07afba2a11a091.png"},{"id":56885075,"identity":"5d48cd30-e905-4dbc-bc76-8a87df8fb7b4","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":251625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolite and gene expression analysis in photocatalytic biohybrids. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of electrons derived from CC NPLs increasing NADH levels and promoting BDO production. (\u003cstrong\u003eb, c\u003c/strong\u003e) OD\u003csub\u003e600\u003c/sub\u003e and BDO yield of XG211 and CC NPLs-XG211 biohybrids under light (5 mW cm\u003csup\u003e-2\u003c/sup\u003e) and dark. Error bar: standard deviation, n = 4. (\u003cstrong\u003ed\u003c/strong\u003e) Measurement of the NADH/NAD\u003csup\u003e+\u003c/sup\u003e and NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratio (5 mW cm\u003csup\u003e-2\u003c/sup\u003e). Error bar: standard deviation, n = 4. \u0026nbsp;(\u003cstrong\u003ee\u003c/strong\u003e) Testing the ATP levels of XG211 and biohybrids under light (5 mW cm\u003csup\u003e-2\u003c/sup\u003e) and dark. Error bar: standard deviation, n = 4. (\u003cstrong\u003ef\u003c/strong\u003e) Intracellular metabolites concentrations in CC NPLs-XG211 biohybrids under light (5 mW cm\u003csup\u003e-2\u003c/sup\u003e) and dark. G6P, Glucose 6-phosphate; FBP, Fructose 1,6-bisphosphate; 3PG, 3-phosphoglycerate; PEP, Phosphoenolpyruvate; AcCoA, Acetyl-CoA; MAL, Malate; 6PGC, 6-phosphogluconate. Error bar: standard deviation, n = 4. (\u003cstrong\u003eg\u003c/strong\u003e) Volcano plot reveals differentially expressed genes (DEGs) in biohybrids under light and dark conditions, showing upregulated and downregulated genes. Blue: Genes down-regulated under light relative to dark conditions. Orange and red: Genes down-regulated under light relative to dark conditions (The red triangles represent genes related to thiamine synthesis); the number of regulated genes indicated in the bracket. (\u003cstrong\u003eh\u003c/strong\u003e) Heatmaps of significantly regulated genes from RNA-seq analysis. P values are determined by a two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/b1425688ea7180ec04d450ce.png"},{"id":56885076,"identity":"c80c55b7-b93c-4e5a-92da-f71d79f534b4","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of electron transfer mechanisms in CC NPLs-XG211 biohybrid systems. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic diagram of NADH regeneration \u003cem\u003ein vitro\u003c/em\u003e by CC NPLs after adding TPP and thiamine.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) NMR analysis was conducted under \u003cem\u003ein vitro\u003c/em\u003e photonic conditions with the addition of TPP or thiamine and CC NPLs catalyzing the conversion of NAD\u003csup\u003e+\u003c/sup\u003e to NADH. (\u003cstrong\u003ec\u003c/strong\u003e) Regeneration of NADH after the addition of TPP for testing \u003cem\u003ein vitro \u003c/em\u003eunder light and dark. Error bar: standard deviation, n = 4. (\u003cstrong\u003ed\u003c/strong\u003e) Schematic diagram of biohybrids and bacteria used to calculate BDO production and carbon yield under light conditions. (\u003cstrong\u003ee, f\u003c/strong\u003e) The ratio in BDO and carbon yield (biohybrid/bacteria) following the addition of varying concentrations of TPP under light. Error bar: standard deviation, n = 4. \u0026nbsp;(\u003cstrong\u003eg\u003c/strong\u003e) Schematic diagram of biohybrids used to calculate BDO and carbon yield under light and dark conditions. (\u003cstrong\u003eh, i\u003c/strong\u003e) The ratio in biohybrid BDO and carbon yield (light/dark) following the addition of varying concentrations of TPP under light and dark conditions. Error bar: standard deviation, n = 4. P values are determined by a two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/06319abfad14bf11897fdf14.png"},{"id":56885313,"identity":"f49bb1de-d754-471f-b9ae-990e542cfdb3","added_by":"auto","created_at":"2024-05-21 18:29:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":119157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssess BDO production and carbon yield in biohybrid/bacteria under light exposure after CRISPR interference gene suppression.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of CRISPR interference gene transcription. (\u003cstrong\u003eb, c\u003c/strong\u003e) The relative increase in biohybrid/bacteria BDO and carbon yield following CRISPRi under light. Cas9, CRISPR-associated protein 9; sgRNA, single guide RNA; dCas9, catalytically inactive version of Cas9; RNAP, RNA polymerase. Error bar: standard deviation, n= 4.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/f2e678388f01acf2e1d4f596.png"},{"id":57773390,"identity":"2501bc30-79cd-4eab-885c-a2dbf67a8f94","added_by":"auto","created_at":"2024-06-05 12:48:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3393467,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/8feb29bf-95ee-4c07-81c5-6c056138cb0d.pdf"},{"id":56885073,"identity":"e008e682-3e13-40d6-a674-dd82226d28f9","added_by":"auto","created_at":"2024-05-21 18:21:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3011140,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials20240516.docx","url":"https://assets-eu.researchsquare.com/files/rs-4431666/v1/35d410e37e7fd11df8521732.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Enhancing Solar-to-Chemical Conversion through Tailoring Dimensions of Semiconductors in Biohybrid Systems","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany chemicals derived from fossil fuels could be sustainably produced via biomanufacturing, reducing the reliance on fossil energy and lowering greenhouse gas emission\u003csup\u003e1, 2\u003c/sup\u003e. However, current biomanufacturing processes primarily reliant on sugar feedstocks suffer from low energy conversion efficiencies and productivities, particularly in the production of highly reduced chemicals\u003csup\u003e3, 4\u003c/sup\u003e. Through the catabolic process, the microbial cell factories release energy nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and adenosine triphosphate (ATP) from sugar, simultaneously generating CO\u003csub\u003e2\u003c/sub\u003e which reduces carbon yields and represents a significant bottleneck in chemical production\u003csup\u003e5\u003c/sup\u003e. Semiconductor-microbe hybrid systems have emerged as a promising solution, integrating efficient light-harvesting nanomaterials with superior whole-cell biocatalysts, allowing the non-photosynthetic bacteria to directly utilize light energy to power the cells\u003csup\u003e6, 7\u003c/sup\u003e. These systems achieve higher carbon yield and productivities by providing additional energy while reducing or eliminating carbon loss\u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLow-dimensional nanomaterials\u003csup\u003e9\u003c/sup\u003e, typically smaller than 100 nm, including zero-dimensional (0D) nanoparticles or quantum dots (QDs), one-dimensional (1D) nanotubes or nanorods (NRs), and two-dimensional (2D) nanosheets or nanoplatelets (NPLs), have been recognized for their cost-effectiveness, enhanced biocompatibility, photocatalytic activity, and stability\u003csup\u003e10-13\u003c/sup\u003e. These nanomaterials dominate in contemporary semiconductor biohybrid systems\u003csup\u003e14, 15\u003c/sup\u003e.\u0026nbsp;To fully harness the potential of those low-dimensional biohybrid systems, it\u0026rsquo;s essential to engineer semiconductors and bacteria based on detailed, systemic, and quantitative insights that enhance energy conversion efficiencies\u003csup\u003e16\u003c/sup\u003e. Although research has begun to explore the variables influencing the effectiveness of semiconductor biohybrids, a comprehensive assessment of nanomaterial dimensions and electron transfer mechanisms is still in its\u0026nbsp;infancy\u003csup\u003e17, 18\u003c/sup\u003e. Current studies have primarily focused on photoelectron transfer mechanisms in biohybrids with extracellular or periplasmic nanomaterial localization\u003csup\u003e19-21\u003c/sup\u003e, where electrons must traverse cellular membranes-an energy-consuming bottleneck\u003csup\u003e22\u003c/sup\u003e. Intracellular nanomaterials, however, allow for the direct generation of photoelectrons within the cytoplasm, facilitating rapid charge transfer\u003csup\u003e23\u003c/sup\u003e. Studies in mammalian and plant cells have emphasized the importance of the dimensional properties for nanomaterials-cell interactions, especially for biomolecule delivery, showing that size and shape can affect cellular internalization and efficiency\u003csup\u003e24-26\u003c/sup\u003e. Yet, the understanding of how nanomaterial dimensions and intracellular photoelectron transfer mechanisms affect solar-driven chemical production in biohybrid systems remains limited, restricting the advancement of efficient solar-driven biomanufacturing.\u003c/p\u003e\n\u003cp\u003eHere we constructed and quantitatively assessed the biohybrid system\u0026rsquo;s performance, focusing on the influence of nanomaterial dimensions on energy conversion efficiency and electron transfer mechanisms. Using low-dimensional cadmium sulfide (CdS) semiconductors (0D QDs, 1D NRs, and 2D NPLs) and 2,3-butanediol (BDO)-producing engineered bacteria \u003cem\u003eVibrio natriegens\u003c/em\u003e, we demonstrated that 2D NPLs (5 monolayers (ML)) exhibit higher internalization rates and photocurrent, leading to increased BDO production (Fig. 1a). To enhance the charge separation efficiency of 5 ML 2D NPLs, we further designed a core/crown (CC) heterostructure. In particular, the CC NPLs structured 2D NPLs biohybrid systems notably enhanced BDO yield by 2.69 times compared to the pure bacterial systems, with a solar-to-chemical efficiency of 2.35%. Electron microscopy and photophysical analysis elucidated a well-integrated 2D NPLs-microbe interface facilitating rapid charge transfer. RNA sequencing (RNA-seq) and metabolomic studies disclosed a novel intracellular photoelectron energy transfer and conversion pathway for generating NADH and ATP, mediated by thiamine pyrophosphate (TPP), resulting in enhanced microbial energy metabolism (Fig. 1b). Our study reported the implementation of 2D nanomaterials in the cytosol as an artificial solar-energy system, and its application in achieving high yield of highly reduced chemicals in the cell factories.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDimensionality-dependent photoelectric conversion and cellular internalization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we first investigated\u0026nbsp;the influence of dimensionality on photoelectric conversion and cellular internalization rates\u0026nbsp;performance using CdS nanocrystals (NCs) with different dimensions. We synthesized 0D QDs, 1D NRs, and 2D NPLs following previously reports\u003csup\u003e27, 28\u003c/sup\u003e. The morphology of these\u0026nbsp;nanomaterials\u0026nbsp;was characterized\u0026nbsp;by transmission electron microscopy (TEM), confirming the dimensions: QDs with a diameter of 4.8 nm, NRs with dimensions of 20\u0026times;5 nm, and 2D NPLs (4 ML) with dimensions of 40\u0026times;40\u0026times;2.2 nm (Fig.\u0026nbsp;2a). UV-visible (UV-vis) spectroscopy revealed maximum absorption peaks at 424 nm, 455 nm, and 424 nm for QDs, NRs, and NPLs, respectively (Supplementary Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhotoelectric conversion efficiency was assessed via current-density-time (i-t) curves measured under blue light (470 nm) illumination. This method reflects the photocatalytic activity of the\u0026nbsp;nanomaterials, a critical parameter in evaluating their performance. Photocurrent measurements under intermittent lighting conditions showed negligible current during \u0026ldquo;dark\u0026rdquo; periods, but significant photocurrent during \u0026ldquo;light\u0026rdquo; periods (Fig.\u0026nbsp;2b). The photocurrent data, as depicted in Fig.\u0026nbsp;2b, c, demonstrated that under blue light, NPLs exhibited a maximum photocurrent of 1.3 \u0026micro;A cm\u003csup\u003e-2\u003c/sup\u003e, which was 3.79 times and 1.96 times higher than that of QDs and NRs, respectively\u0026nbsp;(Fig.\u0026nbsp;2c). This enhancement can be attributed to the extensive 2D surface area and strong 1D quantum confinement in NPLs, which exceed 0D QDs and 1D NRs in promoting rapid charge transfer and effective charge separation\u003csup\u003e29, 30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe next explored the uptake rate of\u0026nbsp;nanomaterials\u0026nbsp;by the bacterial cells, a critical step to achieve semiconductor biohybrid construction\u003csup\u003e22\u003c/sup\u003e. After introducing nanomaterials (1.5 mg L\u003csup\u003e-1\u003c/sup\u003e of each) into a bacterial cell suspension and culturing in minimum medium (detail in the methods), cell growth was monitored by measuring the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e). The growth rates were comparable across all\u0026nbsp;nanomaterial types and a control group without nanomaterials, indicating good biocompatibility of the 1.5 mg L\u003csup\u003e-1\u003c/sup\u003e 0D QDs, 1D NRs, and 2D NPLs\u0026nbsp;with bacterial cells (Supplementary Fig. 2). \u0026nbsp;Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the residual concentration of CdS\u0026nbsp;nanomaterials\u0026nbsp;in the minimum medium after 3 h of culture. Surprisingly, the residual concentrations of 1D NRs and 2D NPLs were notably lower at 20.74 ppb and 13.89 ppb, respectively, compared to 84.11 ppb for 0D QDs, indicating a higher uptake of NPLs by the bacteria (Fig. 2d). We attributed\u0026nbsp;this high penetration rate to the unique flat structure of NPLs, which allowed\u0026nbsp;better access to bacteria due to their ultra-thin longitudinal thickness and adaptability to various bacterial surface topographies under different mechanical conditions\u003csup\u003e31, 32\u003c/sup\u003e. Taking together, we speculated the superior photocurrent and better internalization of 2D NPLs may achieved a higher light-driven chemical production by photosensitizing bacteria.\u003c/p\u003e\n\u003cp\u003eThe atomic-layer thickness of 2D NPLs has been shown to control the optical and electronic properties, such as the\u0026nbsp;carrier dynamics of two-dimensional NPLs\u003csup\u003e33\u003c/sup\u003e, thereby enhancing light absorption and facilitating effective charge separation\u003csup\u003e34\u003c/sup\u003e. Here, we investigated the effect of NPLs\u0026nbsp;thickness (including 3, 4, and 5 ML) on photo-induced\u0026nbsp;current efficiency\u0026nbsp;(Fig.\u0026nbsp;3a,\u0026nbsp;Supplementary Figs. 3 and 4).\u0026nbsp;As shown in Fig.\u0026nbsp;2e, f, the photocurrent of 5\u0026nbsp;ML\u0026nbsp;NPLs was 1.82\u0026nbsp;\u0026micro;A cm\u003csup\u003e-2\u003c/sup\u003e, a 32.43% increase compared to 4\u0026nbsp;ML\u0026nbsp;NPLs and a\u0026nbsp;1.16-fold increase compared to 3\u0026nbsp;ML NPLs, reflecting a decrease in two-dimensional bandgap with increasing thickness, which aids photogenerated electron mobility, particularly in 5 ML NPLs\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;We also investigated the effect of\u0026nbsp;NPLs\u0026nbsp;thickness on internalization efficiency by measuring the residual concentration of Cd\u003csup\u003e2+\u003c/sup\u003e in the minimum medium (Fig. 2g). All thicknesses showed effective absorption, with the lowest residual concentration observed in 5 ML NPLs (8.14 ppb), albeit not significantly different from other thicknesses. Those results indicated the thicker 2D NPLs enhance photoelectrical efficiency while maintain the similar levels of bacterial cells internalization efficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnhanced charge separation efficiency with core/crown heterojunction NPLs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the electron transfer efficiency, as indicated by higher current density, the charge separation efficiency of a nanomaterial is another important factor influencing its photoelectrochemical performance. To improve charge separation efficiency and electron utilization performance, we modulated the energy level distribution by designing and constructing heterojunctions based on 5 ML NPLs (Fig. 3a). In comparison to other heterojunction nanomaterials, core-crown (CC) heterojunctions are capable of achieve efficient electron-hole separation by modulating the energy level distribution without necessitating changes to the thickness of the atomic layers or altering the optical absorption characteristics of the core NPLs. In this regard, zinc selenide (ZnSe) was chosen for its narrow band gap, and a CdS/ZnSe CC NPLs was constructed by epitaxial growth to create a type-II heterostructure.\u003c/p\u003e\n\u003cp\u003eAs shown in\u0026nbsp;Fig. 3b,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTEM images demonstrated the rectangular-shaped morphology of the NPLs with average lateral dimensions of 45\u0026times;45\u0026times;2.2\u0026nbsp;nm.\u0026nbsp;In-situ\u0026nbsp;energy-dispersive X-ray spectroscopy\u0026nbsp;(EDS) mapping (Fig. 3c) provided further evidence for a core/crown composition in the CdS/ZnSe CC NPLs, with CdS (red and green) located in the central region and ZnSe (yellow and blue) at the outer edge.\u0026nbsp;Raman spectroscopy measurements\u0026nbsp;analyzed, the bonding composition of\u0026nbsp;the\u0026nbsp;heterojunction\u0026nbsp;CC NPLs in detail, and\u0026nbsp;identified a\u0026nbsp;characteristic peak for the Cd-S bond at ~302 cm\u003csup\u003e-1\u003c/sup\u003e (longitudinal optics, LO). X-ray diffraction (XRD) spectra demonstrated its crystalline characteristics of the zinc blende phase (Fig. 3e). The UV-vis and photoluminescence (PL) showed spectra shown a strong absorption peak at 440 nm and green emission peak at 510 nm (Fig. 3f). The energy differences between the valence (VB) and conduction (CB) bands reported in Fig. 3g,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere based on UV photoelectron spectroscopy (UPS) measurements and calculations of the absorption and emission energies. The schematic representation in the insert of Fig. 3g\u0026nbsp;described the band-edge alignment for CC NPLs.\u0026nbsp;In the CC NPLs, charges were separated, with the electron wave function localized in the CdS core and the hole in the ZnSe crown. This type-II band alignment drives the photogenerated excitons at the ZnSe crown to migrate towards the core/crown interface, forming a charge-transfer (CT) exciton. The observed broader full width at half maximum (FWHM, 40 nm) was a common feature of type-II nanocrystals\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;Subsequently, we measured the photocurrent of CC NPLs and the residual concentration of Cd\u003csup\u003e2+\u003c/sup\u003e in the minimum medium. As shown in Fig. 2e, f, the photocurrent of CC NPLs is 2.63 \u0026micro;A cm\u003csup\u003e-2\u003c/sup\u003e, increased by 44.51% compared to 5 ML NPLs (1.82\u0026nbsp;\u0026micro;A cm\u003csup\u003e-2\u003c/sup\u003e), which illustrates that the core-crown heterojunctions (CdS/ZnSe CC NPLs) improve the charge separation efficiency. The residual Cd\u003csup\u003e2+\u003c/sup\u003e concentration of CC NPLs (9.71 ppb) after inoculated with bacteria was similar to 5 ML NPLs (8.14 ppb).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImproved bioproduction of \u003cem\u003eV. natriegens\u0026nbsp;\u003c/em\u003evia CC heterojunction NPLs biohybrids \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;natriegens\u003c/em\u003e, recognized as a next-generation host for biotechnology with exceptionally high growth (doubling time of less than 10 minutes) and substrate consumption rate\u003csup\u003e37\u003c/sup\u003e, has demonstrated the capability for extracellular electron transfer which could facilitate the transfer of photo-electrons from semiconductor to bacterial cells in biohybrid systems\u003csup\u003e38\u003c/sup\u003e. The bioproduction of BDO is dependent on the supply of reducing power, thus serving as an excellent platform for evaluating the energy efficiency of biohybrids. Thus, the pET-RABC plasmid\u003csup\u003e39\u003c/sup\u003e, which contains the BDO biosynthetic pathway, was introduced into \u003cem\u003eV. natriegens\u003c/em\u003e, resulting in strain XG211.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that enhanced internalization coupled with higher photocurrent of CC NPLs could improve bacteria photosensitization, we added the\u0026nbsp;nanomaterials to the cell suspension (OD\u003csub\u003e600\u003c/sub\u003e ~ 2.0) of strain XG211 in the minimum medium with 4 g L\u003csup\u003e-1\u003c/sup\u003e glucose, electron sacrificial agent\u0026nbsp;(cystine)\u0026nbsp;and mediator\u0026nbsp;(flavin mononucleotide, FMN). After incubation for 1 h under blue light (470 nm, 5 mW\u0026nbsp;cm\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e), the BDO production of strain XG211 were measured using gas chromatography (GC). We found that BDO production of strain XG211 with\u0026nbsp;CC NPLs\u0026nbsp;nanomaterials\u0026nbsp;under illumination were all higher than their counterpart in absence of illumination or\u0026nbsp;nanomaterials\u0026nbsp;(Fig.\u0026nbsp;4b). Notably, BDO production in the strain with CC NPLs was superior to all other\u0026nbsp;nanomaterials, achieving a final titer of 1.68 g L\u003csup\u003e-1\u003c/sup\u003e (Fig.\u0026nbsp;4b), showing 1.28-fold increase compared with 5 ML NPLs without heterostructure (1.31 g L\u003csup\u003e-1\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, a series of control experiments were conducted to compare the light-driven BDO production with variations in\u0026nbsp;nanomaterial dimensions. Initially, the BDO production of strain with 2D (4 ML) NPLs was 1.05 g L\u003csup\u003e-1\u003c/sup\u003e, showing 35.4% and 36.3% increase when compared to 1D NRs and 0D QDs under illumination, respectively (Fig. 4a).\u0026nbsp;Furthermore, 5 ML NPLs\u0026nbsp;biohybrids\u0026nbsp;achieve higher BDO production compared to 4 ML\u0026nbsp;NPLs biohybrids, while the 3 ML NPLs\u0026nbsp;biohybrids\u0026nbsp;(0.94 g L\u003csup\u003e-1\u003c/sup\u003e) was similar to 4 ML NPLs\u0026nbsp;biohybrids. As aforementioned mentioned, 2D\u0026nbsp;nanomaterials\u0026nbsp;offer better internalization coupled with enhanced photocurrent compared to 0D and 1D\u0026nbsp;nanomaterials, resulting in higher BDO production. This trend is also the same regarding the layers of the\u0026nbsp;nanomaterials. Taking together, our results demonstrated that all low-dimensional CdS\u0026nbsp;nanomaterials\u0026nbsp;were capable of photosensitizing \u003cem\u003eV. natriegens\u003c/em\u003e, with 2D CC NPLs showing the highest efficiency in light-driven chemical production.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;After confirming the BDO production and the superior catalytic performance in 2D CC NPLs the biohybrids, we hypothesize a direct interaction at the nanomaterials-bacteria interface. To this end, we prepared cross-sectional slices of CC NPLs-XG211 biohybrids samples using microtome sections. High angle angular dark field-scanning transmission electron microscopy (HAADF-STEM) image and EDS mapping showed that internalized CC NPLs were composed of Cd, S, Zn and Se, with highly correlated locations (Fig. 4c and Supplementary Fig. 5). Those results clearly revealed the successful transportation of CC NPLs into the cytoplasm of \u003cem\u003eV. natriegens\u003c/em\u003e. Previous studies have reported that intracellular photosensitizers enhanced the efficiency of photoelectron transfer and energy transduction by avoiding the energy loss during photoelectrons transmembrane transfer\u003csup\u003e40, 41\u003c/sup\u003e. We studied the charge-transfer kinetics at this internalized CC NPLs-XG211 biohybrids interface. The photo-excited electrons lifetime and photocurrent of both CC NPLs-XG211 biohybrids and pure CC NPLs were measured (Fig. 4d). The average photogenerated state lifetime of biohybrids was only 0.26 \u0026plusmn; 0.01 ns, showing a 280-fold shorted compared with the CC NPLs alone (72.8 \u0026plusmn; 5.2 ns) (Fig. 4e). Similarly, upon the addition of bacteria, the photocurrent of biohybrid systems decreased by about 69.96% compared to that of CC NPLs alone (Fig. 4f). These results indicated that the assembly of CC NPLs into bacteria results in rapid photo-induced charge transfer between CC NPLs and the bacterial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotoelectron-induced regulation of energy metabolism in biohybrids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe semiconductor harvested light for suppling reducing equivalent instead of sugar oxidation for microbial cells in the biohybrid systems, therefore increasing the carbon yield by reducing/eliminating carbon loss during chemical production (Fig. 5a). We firstly detail analyzed the biomass and glucose-to-BDO of strain XG211 with and without CC NPLs under illumination. The biomass of illuminated biohybrid systems was only slightly higher (~ 28.9% improvement) than all other conditions (Fig. 5b). However, the carbon yield of illuminated biohybrid systems reach 0.457 g g\u003csup\u003e-1\u003c/sup\u003e, showing a 2.69-fold increase compared with XG211 with no\u0026nbsp;nanomaterials, and a 2.01-fold increase compared to its counterpart under dark condition (Fig. 5c).\u0026nbsp;Additionally, the production of acetoin (Fig. 1b), a direct precursor of BDO, was increased by 96.8% under light conditions (Supplementary Fig. 6). The mechanism of this enhanced production yield was further studied as followings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe initially measured the changes of intracellular energy pools including NAD(P)\u003csup\u003e+\u003c/sup\u003e, NAD(P)H, ADP and ATP. The NADH/NAD\u003csup\u003e+\u003c/sup\u003e ratio, represent the redox energy state of cells, in illuminated biohybrid systems surpassed all other conditions, with a ratio of 0.877\u0026nbsp;(Fig. 5d). This ratio indicated a 3.06-fold increase compared with its counterpart with no illumination and a 1.19-fold increase compared with bacterial system in absence of CC NPLs (Fig. 5d). While, the NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratio of illuminated biohybrid systems was\u0026nbsp;23%\u0026nbsp;higher than that of dark condition and was\u0026nbsp;16%\u0026nbsp;higher than that of bacterial system (Fig. 5d). The highest intracellular ATP concentration was also achieved in the illuminated biohybrid systems, increased 24% compared with the dark condition (Fig. 5e). Those results confirmed two-dimensional semiconductor biohybrid systems absorbed light to power the bacterial energy metabolism, thereby promoted the flux of BDO synthetic pathway which required quantity of reducing energy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo reveal the impacts of intracellular CC NPLs on bacterial metabolism during illumination, we performed metabolomics and transcriptomic analysis.\u0026nbsp;We collected biohybrid samples with and without illumination for targeted metabolite quantification analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Central metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP), which\u0026nbsp;were\u0026nbsp;primary sources of reducing power and ATP, were analyzed. As shown in Fig. 5f, the intracellular concentration of key metabolites such as 6-phosphate glucose (G6P), fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3PG) and phosphoenolpyruvate (PEP) in the glycolysis, as well as Acetyl-CoA (AcCoA) and malate (MAL) in the TCA cycle,\u0026nbsp;were\u0026nbsp;increased under light compared to dark condition. Additionally, the concentration of 6-phosphogluconate (6-GPC) in the PPP pathway was also higher under light than dark condition (Fig. 5f). Those results suggested the\u0026nbsp;nanomaterials\u0026nbsp;induced photoelectrons likely improve the activity of central metabolism, which consistence with previous reports\u003csup\u003e19, 42\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo explored global regulation of bacterial cells by illuminated\u0026nbsp;nanomaterials, we further performed RNA-seq to compare gene expression and transcription activation at genome-wide level. Comparing biohybrid with versus without illumination, we identified 28 genes with significantly upregulation and 28 downregulation\u0026nbsp;(log\u003csub\u003e2\u003c/sub\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(fold change (FC))\u0026nbsp;\u0026gt; 1.0 or \u0026lt; -1.0 and P \u0026lt; 0.01) (Fig. 5g,\u0026nbsp;h). Gene ontology (GO) term analysis of the upregulated genes highlighted a common feature: \u0026ldquo;sulfide metabolism\u0026rdquo; or \u0026ldquo;flavin binding\u0026rdquo; (Supplementary Fig. 7), which likely related to electron acceptors or mediators participating in bacterial electron transfer processes. As shown in Fig.\u0026nbsp;5g, h, the upregulated genes including those encoding proteins involved in cellular energy metabolism with illumination, such as the oxidative respiratory chain (Complex Ⅰ [NADH-quinone reductase],\u0026nbsp;Complex Ⅱ [fumarate reductase],\u0026nbsp;Complex Ⅳ [cytochrome ubiquinol oxidase]), FAD [FMN transferase] and RNF [RNF complex, electron transport complex], as well as the ATPase [ATP synthase]. Remarkably, 10 out of 28 upregulated genes were involved in thiamine biosynthesis, for production of TPP (the active form of thiamine), a key cofactor for enzymes involved in central metabolic pathway such as transketolase and dehydrogenase, which participate in PPP, TCA cycle and the link between glycolysis and TCA cycle\u003csup\u003e43-45\u003c/sup\u003e. Those enzymes are vital for the production of NADH, NADPH and ATP. The above up-regulation\u0026nbsp;genes\u0026nbsp;were further confirmed by the real-time fluorescence quantitative PCR (qRT-PCR) results, which consistence with the RNA-seq results (Supplementary Fig. 8).\u003c/p\u003e\n\u003cp\u003eTaking together, those results suggesting the illuminated biohybrid systems not only provide extra-energy from light but also promote both carbon metabolism and energy metabolism of bacteria, notably increasing carbon yield by reducing or eliminating carbon loss during chemical production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification and application of photoelectron-induced regulation in biohybrids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our biohybrid systems, semiconductors capture light to supply reducing equivalents for cellular metabolism, significantly enhancing the thiamine/TPP synthetic pathway. This pathway typically boosts the native energy machinery (central metabolism) to generate more reducing power. Observations of these effects led us to hypothesize that converting photoelectrons into reducing power might require the mediation of TPP. To explore this hypothesis, we analyzed the rate of NADH generation from CC NPLs with and without TPP under \u003cem\u003ein vitro\u003c/em\u003e conditions (Fig.\u0026nbsp;6a,\u0026nbsp;b). In a typical experiment, 1.5 mg mL\u003csup\u003e-1\u003c/sup\u003e CC NPLs, 1 mM NAD\u003csup\u003e+\u003c/sup\u003e with or without TPP was inoculated under\u0026nbsp;470\u0026nbsp;nm light (5 mW cm\u003csup\u003e-2\u003c/sup\u003e). NADH regeneration was monitored by the absorption at 340 nm (Abs\u0026nbsp;340\u0026nbsp;nm) and the final NADH product was confirmed by nuclear magnetic resonance (NMR, Fig.\u0026nbsp;6b). Adding TPP resulted in a 4.45-fold increase in NADH production under light conditions compared to controls without TPP, whereas NADH production remained unchanged under dark conditions\u0026nbsp;(Fig.\u0026nbsp;6c).\u0026nbsp;These findings were corroborated by NMR, affirming that the increase in Abs 340 nm was indeed attributable to heightened NADH production (Fig.\u0026nbsp;6b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, the presence of thiamine (vitamin B1), commonly used as a substrate in cultures for TPP biosynthesis, led to a 9.45-fold increase in NADH production under light conditions, with no improvement noted under dark conditions (Supplementary Fig. 9). Based on these results and corresponding \u003cem\u003ein vitro\u003c/em\u003e experiments, both TPP and thiamine not only enhance reducing power production from photoelectrons but also boost the native central metabolism. Recent studies also suggest that TPP/thiamine promotes NADH production and electron transfer in two bacterial species co-cultures\u003csup\u003e46\u003c/sup\u003e. We speculated the photoelectron transfer and solar-to-chemical efficiency in biohybrid systems might enhanced by the addition of TPP/thiamine.\u003c/p\u003e\n\u003cp\u003eTo investigate further, we cultured biohybrid systems with varying concentrations (0, 0.3, 0.6, 1, 3, 6 mM) of TPP or thiamine, analyzing the ratio of BDO production and carbon yield of illuminated biohybrids compared to control bacteria (Fig. 6d-6f), and also compared biohybrid performance under light versus dark conditions (Fig. 6g-6i). The ratio of BDO production and carbon yield of illuminated biohybrids increased by 11.73% and 8.57% respectively with 0.6 mM TPP compared to the conditions without TPP, showing a decrease when TPP concentrations exceeded 1 mM (Fig. 6e, f). Similarly, the ratio of BDO production and carbon yield of the biohybrid systems under light increased by 22.75% and 24.02% with 0.6 mM TPP compared to conditions without TPP, decreasing with higher TPP concentrations (Fig. 6h, i). However, the growth of biomass was increased 38.09% and 21.29% with 6 mM TPP under light and dark compared to the control without TPP, respectively (Supplementary Fig. 10). Similar results were observed with thiamine, where both BDO production and carbon yield increased with 0.6 mM thiamine (Supplementary Fig. 11). These results indicate that TPP/thiamine indeed promotes solar-to-chemical efficiency, but the concentration is critical for optimizing carbon partitioning between desired chemicals and biomass growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification and application of photoelectron-induced regulation in biohybrids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our biohybrid systems, semiconductors capture light to supply reducing equivalents for cellular metabolism, significantly enhancing the thiamine/TPP synthetic pathway. This pathway typically boosts the native energy machinery (central metabolism) to generate more reducing power. Observations of these effects led us to hypothesize that converting photoelectrons into reducing power might require the mediation of TPP. To explore this hypothesis, we analyzed the rate of NADH generation from CC NPLs with and without TPP under \u003cem\u003ein vitro\u003c/em\u003e conditions (Fig.\u0026nbsp;6a,\u0026nbsp;b). In a typical experiment, 1.5 mg mL\u003csup\u003e-1\u003c/sup\u003e CC NPLs, 1 mM NAD\u003csup\u003e+\u003c/sup\u003e with or without TPP was inoculated under\u0026nbsp;470\u0026nbsp;nm light (5 mW cm\u003csup\u003e-2\u003c/sup\u003e). NADH regeneration was monitored by the absorption at 340 nm (Abs\u0026nbsp;340\u0026nbsp;nm) and the final NADH product was confirmed by nuclear magnetic resonance (NMR, Fig.\u0026nbsp;6b). Adding TPP resulted in a 4.45-fold increase in NADH production under light conditions compared to controls without TPP, whereas NADH production remained unchanged under dark conditions\u0026nbsp;(Fig.\u0026nbsp;6c).\u0026nbsp;These findings were corroborated by NMR, affirming that the increase in Abs 340 nm was indeed attributable to heightened NADH production (Fig.\u0026nbsp;6b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, the presence of thiamine (vitamin B1), commonly used as a substrate in cultures for TPP biosynthesis, led to a 9.45-fold increase in NADH production under light conditions, with no improvement noted under dark conditions (Supplementary Fig. 9). Based on these results and corresponding \u003cem\u003ein vitro\u003c/em\u003e experiments, both TPP and thiamine not only enhance reducing power production from photoelectrons but also boost the native central metabolism. Recent studies also suggest that TPP/thiamine promotes NADH production and electron transfer in two bacterial species co-cultures\u003csup\u003e46\u003c/sup\u003e. We speculated the photoelectron transfer and solar-to-chemical efficiency in biohybrid systems might enhanced by the addition of TPP/thiamine.\u003c/p\u003e\n\u003cp\u003eTo investigate further, we cultured biohybrid systems with varying concentrations (0, 0.3, 0.6, 1, 3, 6 mM) of TPP or thiamine, analyzing the ratio of BDO production and carbon yield of illuminated biohybrids compared to control bacteria (Fig. 6d-6f), and also compared biohybrid performance under light versus dark conditions (Fig. 6g-6i). The ratio of BDO production and carbon yield of illuminated biohybrids increased by 11.73% and 8.57% respectively with 0.6 mM TPP compared to the conditions without TPP, showing a decrease when TPP concentrations exceeded 1 mM (Fig. 6e, f). Similarly, the ratio of BDO production and carbon yield of the biohybrid systems under light increased by 22.75% and 24.02% with 0.6 mM TPP compared to conditions without TPP, decreasing with higher TPP concentrations (Fig. 6h, i). However, the growth of biomass was increased 38.09% and 21.29% with 6 mM TPP under light and dark compared to the control without TPP, respectively (Supplementary Fig. 10). Similar results were observed with thiamine, where both BDO production and carbon yield increased with 0.6 mM thiamine (Supplementary Fig. 11). These results indicate that TPP/thiamine indeed promotes solar-to-chemical efficiency, but the concentration is critical for optimizing carbon partitioning between desired chemicals and biomass growth.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we report a 2D nanomaterials-biohybrid systems that achieve higher carbon yield and solar-to-chemical efficiency. Through the systematic exploration, our findings reveal that 2D CC NPLs nanomaterials exhibit superior photoelectrochemical efficiency and internalization rates compared to 0D and 1D nanomaterials, consequently yielding the highest production yield of chemical. The intracellular 2D nanomaterials offer a closed nanomaterial-microbe interaction, generate photoelectrons in the cytosol, thereby bypassing energy-consuming cross membrane electron transfer and facilitating faster electron conversion. Unlike previous studies focused on extracellular or periplasmic electron transfer pathways (Supplementary Table\u0026nbsp;2), our research unveils a novel intracellular photoelectron energy conversion pathway. Transcriptomic analysis has shown an upregulation of the TPP biosynthetic pathway and the oxidative respiratory chain. Moreover, \u003cem\u003ein vitro\u003c/em\u003e assays have demonstrated TPP promotes NADH generation, suggesting TPP mediates the transfer and conversion of photoelectron energy from the nanomaterials to NADH and ATP. Those findings will guide the rational engineering of semiconductor biohybrid systems to approach the maximum solar-to-chemical efficiency during bioproduction process.\u003c/p\u003e\u003cp\u003eTPP is a key cofactor for enzymes involved in central metabolic pathways such as glycolysis, pentose-phosphate pathway and the citric acid cycle, all vital for the bioproduction of ATP and NAD(P)H\u003csup\u003e47\u003c/sup\u003e, thus it’s essential to all life. Here, we discovered that TPP also plays a role in artificial photoelectron energy conversion. Future detailed studies should explore the mechanisms of TPP-mediated photoelectron transfer, such as the number of electrons each TPP molecule can carry, and how native catabolic processes are cross-regulated with photoelectrons to provide energy and metabolic precursors for chemical production. Our preliminary data indicate that varying TPP concentrations differently regulate BDO production and biomass growth in the light-driven semiconductor biohybrid: lower TPP concentrations enhance BDO production, whereas higher concentrations promote biomass growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, h and Supplementary Fig.\u0026nbsp;10). Those results suggest TPP plays a key role in partitioning carbon flux towards biomass and desired chemicals. Moving forward, we need to understand the regulatory mechanisms of TPP on carbon and energy metabolism to achieve the maximum chemical productivity and carbon utilization efficiency, ultimately leading a higher solar-to-chemical conversion efficiency.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSynthesis and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003cstrong\u003eurification of various nanocrystals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCdS QDs.\u0026nbsp;\u003c/strong\u003eCdS QDs were prepared according to the reported procedure\u003csup\u003e48, 49\u003c/sup\u003e. Cd-precursor:\u0026nbsp;a\u0026nbsp;mixture of 0.256 g CdO, 6.3 mL oleic acid and 13.7 mL ODE was heated in a round-bottomed flask at 250\u0026nbsp;\u0026deg;C\u0026nbsp;under N\u003csub\u003e2\u003c/sub\u003e flow until the mixture became clear. S-precursor:\u0026nbsp;a\u0026nbsp;mixture of 64 mg of S and 20 mL of ODE was heated in a round-bottomed flask at 180 \u0026deg;C until the mixture became clear. The mixture of 4 mL of Cd-precursor, 6 mL of ODE was bubbled with N\u003csub\u003e2\u003c/sub\u003e, then heated to 265\u0026nbsp;\u0026deg;C\u0026nbsp;from room temperature. 2 mL of S-precursor was injected into the mixture and the temperature was reduced to 220\u0026nbsp;\u0026deg;C\u0026nbsp;and held at this temperature for 100 seconds. The reaction was then carried out at 220\u0026nbsp;\u0026deg;C\u0026nbsp;for 1 minute with the addition of 1 mL of S-precursor and 1 mL of Cd-precursor. By repeating this process, the QDs can be brought to the desired size (2 times for CdS QDs with diameter of 4.8 nm)\u0026nbsp;and then the mixture is cooled to room temperature to stop the reaction. After the reaction, the liquid mixture was transferred to a centrifuge tube and acetone (three times the volume of the reaction mixture) was added to precipitate the QDs. The mixture was shaken well and centrifuged at 5,000 rpm for 10 minutes. The supernatant was discarded and the precipitate redispersed in hexane. A threefold excess of methanol was added, the mixture was shaken well and centrifuged at 5,000 rpm for 10 minutes. Repeat the above purification and store in hexane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCdS rods (50.0\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026times;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5.0\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e0.3 nm).\u0026nbsp;\u003c/strong\u003eCdS rods\u0026nbsp;samples were synthesized using a previously reported procedure\u003csup\u003e28, 50\u003c/sup\u003e. For\u0026nbsp;the\u0026nbsp;reaction, a 25 mL 3-necked flask was filled with 210 mg of CdO and 2.75 g of TOPO.\u0026nbsp;The\u0026nbsp;0.8 g ODPA and 0.22 g TDPA were used. The mixture was evacuated at 120\u0026nbsp;\u0026deg;C\u0026nbsp;for 30 minutes and then heated to 320\u0026nbsp;\u0026deg;C\u0026nbsp;under N\u003csub\u003e2\u003c/sub\u003e for 15 minutes to allow complexation of cadmium with phosphonic acid. To remove water formed during complexation, the reaction mixtures were cooled to 120\u0026nbsp;\u0026deg;C\u0026nbsp;and evacuated again for 1 h. While reheating to 320\u0026nbsp;\u0026deg;C, 2 g of TOP was injected into each flask.\u0026nbsp;1.3 g\u0026nbsp;TOP-S (TOP and elemental S in a 1:1 molar ratio) was then injected and the nanocrystals were grown at 315\u0026nbsp;\u0026deg;C\u0026nbsp;for 85 minutes. After cooling, the nanorods were washed several times by adding equal amounts of nonanoic acid and isopropanol, followed by centrifugation to precipitate the CdS nanorods. The supernatant was removed, and the precipitated nanorods were redispersed in fresh toluene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3 ML NPLs.\u0026nbsp;\u003c/strong\u003eThe CdS NPLs were synthesized according to the reported procedure\u003csup\u003e51\u003c/sup\u003e. The S-ODE solution was prepared by dissolution of 0.0320 g of sulfur (1.0 mmol) in 20 g of ODE by gentle sonication and stored in a closed vial. Cadmium acetate dihydrate (0.1066 g, 0.4 mmol), S-ODE (2.0032 g, 0.1 mmol S), myristic acid (0.0914 g, 0.4 mmol) and 6.0 g ODE were bubbled with N\u003csub\u003e2\u003c/sub\u003e for 10 minutes, then heated from room temperature to 180 \u0026deg;C in 10 minutes and kept under N\u003csub\u003e2\u003c/sub\u003e flow for a further 30 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4 ML NPLs.\u0026nbsp;\u003c/strong\u003eCadmium acetate dihydrate (0.1066 g, 0.4 mmol), S-ODE (2.0032 g, 0.1 mmol S), oleic acid (0.113 g, 0.4 mmol) and 6.0 g ODE were bubbled with N\u003csub\u003e2\u003c/sub\u003e for 10 minutes. After switching to N\u003csub\u003e2\u003c/sub\u003e protection, the solution was heated from room temperature to 260 \u0026deg;C in 15 minutes and held at 260 \u0026deg;C for 1 minute.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5 ML NPLs.\u0026nbsp;\u003c/strong\u003eCdO and stearic acid (3 times the molar ratio of CdO) were first bubbled with N\u003csub\u003e2\u003c/sub\u003e for 30 minutes, then heated to 160 \u0026deg;C and kept at this temperature until the color of the mixture became almost transparent after about 2 h of reaction. During the cooling process, the solution was added to a large beaker of hot acetone, kept at 55~60 \u0026deg;C, and stirred vigorously for about 10~15 minutes. The white precipitates were separated from the supernatant by timely vacuum filtration. The purification procedure was repeated four more times before the precipitates (Cd(St)\u003csub\u003e2\u003c/sub\u003e) were placed in a vacuum oven for overnight pumping treatments at room temperature. Cd(St)\u003csub\u003e2\u003c/sub\u003e (0.1356 g, 0.2 mmol), S-ODE (2.0032 g, 0.1 mmol S) and 6.0 g ODE were bubbled with N\u003csub\u003e2\u003c/sub\u003e for 10 minutes, then heated from room temperature to 190 \u0026deg;C within 8 minutes and then held at the specified temperature under N\u003csub\u003e2\u003c/sub\u003e flow for 8 minutes. After cooling to room temperature, 0.1066 g of cadmium acetate dihydrate (0.4 mmol) was added, the solution was bubbled with N\u003csub\u003e2\u003c/sub\u003e for 10 minutes and then heated from room temperature to 250 \u0026deg;C in 15 minutes and held at 250 \u0026deg;C for a further 1 minute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of CdS NPLs of various thicknesses.\u003c/strong\u003e The CdS NPLs were mixed with chloroform and oleylamine (10% volume ratio of oleylamine) to form a clear solution. A mixture of acetone and methanol (30% volume ratio of methanol and 5 times volume of chloroform/oleylamine) was then added, and after sonication for 1 minute, the solution was centrifuged at 11,000 rpm for 5 minutes. The supernatant was discarded and the precipitates were collected for a further three rounds of the above purification and redispersed in fresh toluene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCC NPLs.\u0026nbsp;\u003c/strong\u003eCC NPLs were synthesized following a method from the literature with slight modifications. The TOP-Se (1 M) solution was prepared by dissolving selenium in TOP with vigorous stirring and stored in a sealed vial under N\u003csub\u003e2\u003c/sub\u003e. 5 ML\u0026nbsp;NPLs dissolved in 5 mL ODE, 238 \u0026mu;L (0.75 mmol) oleic acid and 109.7 mg (0.5 mmol) zinc acetate were placed in a 25 mL four-necked round bottom flask and degassed under vacuum for 60 minutes at room temperature. The reaction mixture was heated to 255 \u0026deg;C under N\u003csub\u003e2\u003c/sub\u003e flow and after reaching this temperature, 50 \u0026mu;L of 1 M TOP-Se (0.05 mmol Se) in 1 mL ODE was rapidly injected into the reaction mixture. Allow to react for 20 minutes, then cool to room temperature in a water bath. The product is dispersed in hexane and then purified with ethanol by two rounds of centrifugation and finally redispersed in toluene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhase transfer of these CdS nanocrystals to water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phase transfer of the sample is carried out according to the reported ligand exchange method with slight modifications\u003csup\u003e52\u003c/sup\u003e. In a typical procedure, 1 mL of nanocrystals dispersion in toluene (around\u0026nbsp;5 mg mL\u003csup\u003e-1\u003c/sup\u003e) was combined with 0.2 mL DMF solution of NOBF\u003csub\u003e4\u003c/sub\u003e (10 mg\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e) at room temperature. The resulting mixture was gently shaken until the precipitation of the nanocrystals could be observed, which was usually within 5 minutes. After centrifugation to remove the supernatant, the precipitated nanocrystals can be redispersed in the hydrophilic media DMF. To remove the excess ligands, DMF and toluene (1:1 v/v) were added to flocculate the nanocrystals dispersion. After centrifugation, the nanocrystals can be redispersed by adding the polar solvents mentioned above to form a stable colloidal dispersion. The hydrophilic nanocrystals obtained by NOBF\u003csub\u003e4\u003c/sub\u003e treatment can be further functionalized by other capping molecules through secondary ligand exchange reactions, achieving a complete phase transfer of nanocrystals between hydrophobic and hydrophilic. The NOBF\u003csub\u003e4\u003c/sub\u003e-treated nanocrystals were then redispersed in 1 mL\u0026nbsp;of an aqueous solution of L-cysteine\u0026nbsp;(1 mg\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e, adjusted to pH \u0026gt; 10 with NaOH). Subsequent shaking (overnight) allowed the L-\u0026nbsp;cysteine\u0026nbsp;to successfully bind to the surface of the nanocrystals. The nanocrystals were precipitated by the addition of ethanol. The precipitated nanocrystals were redispersed in water to form a stable colloidal dispersion after centrifugation to remove the supernatant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of nanocrystals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll measurements were carried out at room temperature. The absorption spectra of the nanocrystals were recorded using a Carry 5000 (Agilent). The PL, PLE and lifetime were measured on a HORIBA FL-3 3D fluorescence spectrometer. Raman scattering information from the sample was collected using a laser confocal Raman spectrometer (Renishaw InviA-Reflex). A JEOL JEM-2800 transmission electron microscope (TEM) was employed for TEM,\u0026nbsp;scanning transmission electron microscopy\u0026nbsp;(STEM)\u0026nbsp;and energy-dispersive X-ray spectroscopy\u0026nbsp;(EDS)\u0026nbsp;mapping investigations. A Bruker D8 Advance diffractometer equipped with Ni-filtered Cu K\u0026alpha; radiation was used to collect the powder X-ray diffraction\u0026nbsp;(XRD)\u0026nbsp;data. For\u0026nbsp;powder X-Ray\u0026nbsp;diffraction\u0026nbsp;(PXRD), samples are prepared by evaporating the solvent directly from the concentrated dispersion onto a single crystal silicon sample holder. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture medium and condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(1) Rich culture medium\u003c/strong\u003e \u003cstrong\u003eLBv2 (per 1 L)\u003c/strong\u003e: 25 g LB powder, 11.9 g NaCl, 0.313 g KCl, 2.2 g MgCl\u003csub\u003e2\u003c/sub\u003e, and all solidified media for bacterial growth contain 1.5% [w/v] agar.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(2)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVN\u003c/strong\u003e \u003cstrong\u003eminimum medium\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(per 1 L)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(pH=7.5)\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e5\u0026nbsp;g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 15\u0026nbsp;g NaCl, 1\u0026nbsp;g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1\u0026nbsp;g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.25\u0026nbsp;g MgSO\u003csub\u003e4\u003c/sub\u003e, 0.036\u0026nbsp;g CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 16.4\u0026nbsp;mg FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 4\u0026nbsp;g glucose, 10\u0026nbsp;mg MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 0.3\u0026nbsp;mg CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 1\u0026nbsp;mg ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.02\u0026nbsp;mg NiCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eV. natriegens\u003c/em\u003e cells were cultured overnight in LBv2 media containing appropriate antibiotics (kanamycin 200 \u0026mu;g\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e and\u0026nbsp;chloramphenicol 30\u0026nbsp;\u0026mu;g\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e). For the main culture, 100 mL of LBv2 medium was inoculated with 1 mL of the above preculture and cultured for the desired time (3 h) at 37 \u0026deg;C with agitation at 220 rpm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe BDO producing strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe electrocompetent cells of \u003cem\u003eV. natriegens\u003c/em\u003e cells were prepared as described previously\u003csup\u003e42\u003c/sup\u003e. The pET-RABC plasmid was introduced via electroporation. Gently mix 200 ng of plasmid pET-RABC with 100 \u0026micro;L of electrocompetent cells in a chilled 1.5 mL microcentrifuge tube. Transfer the cell-DNA suspension to a cooled electroporation cuvette with a 0.2 cm gap. Electroporate cells at 1.7 kV and immediately recover them in 500 \u0026mu;L of recovery medium (BHI supplemented with v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 680 mM sucrose)). Incubate the cells at 37 \u0026deg;C for 1 h, then plate them on agar plates containing appropriate antibiotics. Incubate the plates at 37 \u0026deg;C overnight for colony growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ebiohybrid systems\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and BDO production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;strain\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eXG211\u0026nbsp;was\u0026nbsp;inoculated in VN medium supplemented with 1.5\u0026nbsp;mg\u0026nbsp;L\u003csup\u003e-1\u003c/sup\u003e CdS, and the OD\u003csub\u003e600\u003c/sub\u003e was adjusted to 0.1. The aerobic cultivation was conducted in an incubator with the temperature set to 37 \u0026deg;C. The cells were collected after 3 h, washed once with VN medium and adjusted to OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof ~2.0. Add 1\u0026nbsp;mM cysteine as electron sacrificial agent and 5\u0026nbsp;\u0026micro;M flavin mononucleotide\u0026nbsp;(FMN) as mediator. React in 5 mW\u0026nbsp;cm\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e blue light (470 nm) for 1 h at 37 \u0026deg;C with agitation at 200 rpm, and take the supernatant for GC test, collect bacteria for metabolite test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotoelectrochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UV-vis (Carry 5000 (Agilent)) was used to measure the direct bandgap. The dispersed nanoparticles or semiconductor biohybrids were loaded on carbon paper, respectively, to form a uniform 1 \u0026times; 1 cm\u003csup\u003e2\u003c/sup\u003e film by drop casting and vacuum drying. The photoelectrochemical measurements were performed by a standard three-electrode configuration in electrolyte (PBS). Ag/AgCl (3 M NaCl) and Pt wire served as the reference and counter electrode, respectively. A 470 nm blue light panel was employed as the laser, with the light intensity calibrated to 5 mW\u0026nbsp;cm\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e. Current densities were recorded under 0.5 V bias vs Ag/AgCl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasuring the remaining Cd\u003csup\u003e2+\u003c/sup\u003e content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe XG211 strain was cultured in a rich culture medium at 37\u0026nbsp;\u0026deg;C for approximately 3 h. Subsequently, 1% of the bacterial culture was transferred into fresh VN medium and grown at 37\u0026nbsp;\u0026deg;C for an additional 3 h. The initial optical density at OD\u003csub\u003e600\u003c/sub\u003e being\u0026nbsp;approximately 0.1. During this culture period, three different types of nanomaterials, namely 0D QDs, 1D NRs, and 2D NPLs, were separately added to the\u0026nbsp;VN\u0026nbsp;medium at a concentration of 1.5 mg\u0026nbsp;L\u003csup\u003e-1\u003c/sup\u003e. The supernatants were collected to measure the removal efficiency using ICP-MS (Inductively Coupled Plasma Mass Spectrometry) with a NexION 1000 instrument from PerkinElmer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantifying intracellular metabolites in\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;biohybrids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify intracellular metabolites, the extraction method was adapted from Gao \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e42\u003c/sup\u003e. Specifically, cells were collected by centrifugation at 13,000 rpm for 10 minutes at 4\u0026nbsp;\u0026deg;C\u0026nbsp;and immediately resuspended in 1 mL of methanol/water (80:20, vol/vol) pre-cooled at \u0026ndash;80\u0026nbsp;\u0026deg;C. The samples were centrifuged at 13,000 rpm for 10 minutes at 4\u0026nbsp;\u0026deg;C. The supernatants\u0026nbsp;were collected for quantification of metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS, SCIEX Triple Quad\u0026trade; 5000+ QTRAP\u0026reg; Ready, AB SCIEX, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantifying BDO and glucose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the BDO concentration, the bacterial culture was centrifuged at 12,000 rpm for 10 minutes, and the same volume of supernatant and isometric ethyl acetate was mixed for sonication for 20 minutes and centrifuged for 10 minutes. Supernatant was filtered using nylon membrane (0.22 \u0026mu;m) and then determined using gas chromatography (GC). The GC system (Agilent 8890, Agilent Technologies Inc., USA) is equipped with a DB-WAX capillary column (30 m \u0026times; 0.53 mm \u0026times; 1 \u0026mu;m) and a flame ionization detector. Hydrogen gas was used as the carrier gas. The injector and detector temperature were maintained at 250\u0026nbsp;\u0026deg;C, and the oven temperature was 80\u0026nbsp;\u0026deg;C. The injection volume was 1 \u0026mu;L. To obtain the calibration curves for quantifying BDO and acetoin, a series of standards were quantified by GC system after the same pretreatment. Glucose content was determined using glucose assay reagent (Beyotime, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracellular NADH regeneration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 1 mL of VN medium containing 1 mM NAD\u003csup\u003e+\u003c/sup\u003e, 1.5 mg\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e of CC NPLs, 1 mM cysteine as a sacrificial agent, and 5 \u0026mu;M FMN as a mediator were added. After a 2\u0026nbsp;h reaction, the absorbance at 340 nm in the test solution was measured to calculate the amount of NADH generated. The impact of adding 0.06 mM\u0026nbsp;thiamine or 1 mM TPP to the above solution on NADH production was observed. Nuclear magnetic resonance (NMR) was employed for the quantification of NADH content in the test solution. The test solution comprised 450 \u0026mu;L of sample solution and 50 \u0026mu;L of D\u003csub\u003e2\u003c/sub\u003eO. NMR data were acquired using either the Bruker AVANCE NEO 400 or AVANCE III HD50 spectrometer. The collected data were processed and integrated using Bruker TopSpin software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of quantum efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe calculation methods of quantum yield (QE) were mostly applicable to monochromatic light or single wavelength\u003csup\u003e22\u003c/sup\u003e, such as\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58854_b38fc7f3db2c487f/58854_custom_files/img1716315380.png\" width=\"415\" height=\"64\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eN\u003csub\u003ea\u003c/sub\u003e\u003c/em\u003e is \u0026nbsp;Avogadro\u0026rsquo;s constant (6.02\u0026times;10\u003csup\u003e23\u003c/sup\u003e) , C\u003cem\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/em\u003e represents the difference in the molar amount of BDO production between biohybrid systems and non-hybrid systems. V=total suspension vol (1 mL). \u0026Phi;\u003csub\u003eph\u003c/sub\u003e is the measured average photon flux, \u0026nbsp;\u0026Phi;\u003csub\u003eph\u003c/sub\u003e cm\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e= 1.18\u0026times;10\u003csup\u003e14\u003c/sup\u003e cm\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. t=reaction time (3600 s). A=area of illumination (1.4 cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eAcetoin+NADH+H\u003csup\u003e+\u003c/sup\u003e\u0026rarr;\u0026nbsp;BDO+NAD\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e+2e\u003csup\u003e-\u003c/sup\u003e+H\u003csup\u003e+\u003c/sup\u003e\u0026rarr;NADH\u003c/p\u003e\n\u003cp\u003eAcetoin+2e\u003csup\u003e-\u003c/sup\u003e+H\u003csup\u003e+\u003c/sup\u003e\u0026rarr;BDO\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58854_b38fc7f3db2c487f/58854_custom_files/img171631538045.png\" width=\"474\" height=\"70\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eRNA seq\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCC NPLs-XG211 biohybrids were cultured in VN medium under both light and dark conditions for 1 h. Subsequently, the harvested cells were collected by centrifugation (4,000\u0026nbsp;rpm, 4 \u0026deg;C, 20 minutes) and flash-frozen in liquid nitrogen. RNA-seq experiments were conducted by Sangon Biotech (Shanghai) Co., Ltd. All data analyses were performed on the cloud platform of Sangon Biotech (Shanghai) Co., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003eeal-time fluorescence quantitative PCR\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 1 h of light and dark treatment, the hybrid systems were collected and immediately snap-frozen in liquid nitrogen. RNA samples were extracted using the Ultrapure RNA Kit (Vazyme Biotech Co., Ltd., China), and genomic DNA was eliminated with DNase I (Vazyme Biotech Co., Ltd., China). Subsequently, qRT-PCR was performed on a qTOWER3 thermal cycler (Analytik Jena, Germany) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China). The relative transcript levels of each gene were normalized to the 16S rRNA gene (\u003cem\u003ePN96_00205\u003c/em\u003e, an internal control) of \u003cem\u003eV. natriegens\u0026nbsp;\u003c/em\u003eusing the primers in Supplementary Table 1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiew, F.E., \u003cem\u003eet al.\u003c/em\u003e Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale. Nat. Biotech. 40, 335\u0026ndash;344 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScown, C.D., Keasling, J.D. Sustainable manufacturing with synthetic biology. Nat. Biotech. 40, 304\u0026ndash;307 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Z., Wang, K., Chen, Y., Tan, T., Nielsen, J. 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Soc. 133, 998\u0026ndash;1006 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to Prof. Ping Xu\u0026rsquo;s group for their generous provision of the pET-RABC vector containing BDO biosynthetic genes. We acknowledge the Shenzhen Infrastructure for Synthetic Biology for instrument support and technical assistance. This work was supported by the National Natural Science Foundation of China (Grant No.\u0026nbsp;32230060, 32171426,\u0026nbsp;22171132), the National Key R\u0026amp;D Program of China (Grant No. 2021YFA0910800, 2021YFA0909900,\u0026nbsp;2022YFC3401802), Shenzhen Science and Technology Program (Grant No.\u0026nbsp;JCYJ20220818101804010, RCYX20221008092901004, ZDSYS20220606100606013, KJZD20230923114419039 and JCYJ20220531100006011) and the Natural Science Foundation of Guangdong Province,\u0026nbsp;the Science and Technology Program in Jiangsu Province (BK20232041), the Program for Innovative Talents and Entrepreneurs in Jiangsu (020513006012 and 020513006014) and the Zijin Scholars Foundation (0205181022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was conceptualized by\u0026nbsp;Y. W.\u0026nbsp;and X.G., and\u0026nbsp;was supervised\u0026nbsp;C. Y., X. G., Y. W.. X. K. prepared the materials and performed the characterization. M. G. performed the experiments of the biohybrid construction and characterization. W. C. performed the metabolomics. W. Y. and X. Y. performed the experiment of CRISPRi. X. W. and S. P. performed the experiment of photocurrent measurement. M. G., X. K., Y. W., and X. G. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in this manuscript are available in the paper and its Supplementary Information.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4431666/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4431666/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntegrating light-harvesting semiconductor materials with biocatalysts offers a promising approach for solar-power production of fuels and fine chemicals. Despite significant advances, the influence of materials’ dimensions on energy utilization efficiency and the involved photoelectron transfer pathways remains largely to be explored. Here, we investigated the effect of dimensionality on the energy conversion efficiency in semiconductor nanomaterial-based biohybrid systems. We found that the intracellularly localized 2D nanoplatelets, particularly with core-crown heterostructures, were more efficient in supplying energy for microbial chemical production than the lower-dimensional nanomaterials. The biohybrids possessing the 2D nanoplatelets exhibited a 2.69-fold increase in 2,3-butanediol (BDO) production yield and achieved 2.35% solar-to-chemical conversion efficiency. Based on metabolomic and transcriptomic analyses, we identified a novel thiamine pyrophosphate (TPP)-mediated pathway of energy generation from photoexcited electrons. Furthermore, the addition of TPP enhanced the BDO production of the biohybrids under illumination. Our results demonstrate the potential to increase the solar-to-chemical conversion efficiency of semiconductor biohybrids by tailoring the dimension of semiconductor nanomaterials and engineering the intracellular electron transfer and energy generation pathways.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*\u003c/strong\u003eMingming Guo and Xinke Kong contributed equally to this work.\u003c/p\u003e","manuscriptTitle":"Enhancing Solar-to-Chemical Conversion through Tailoring Dimensions of Semiconductors in Biohybrid Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-21 18:20:55","doi":"10.21203/rs.3.rs-4431666/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1bca7838-d3a0-4e9f-a58a-26a2ef80bf80","owner":[],"postedDate":"May 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32012218,"name":"Biological sciences/Biotechnology/Metabolic engineering"},{"id":32012219,"name":"Biological sciences/Biochemistry/Biocatalysis"},{"id":32012220,"name":"Physical sciences/Materials science/Materials for energy and catalysis/Photocatalysis"}],"tags":[],"updatedAt":"2024-06-27T12:30:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-21 18:20:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4431666","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4431666","identity":"rs-4431666","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}