Polymer semiconductor films and bacteria hybrid artificial bio-leaves

Abstract

Bio-artificial photosynthetic systems can reduce CO₂ into multicarbon compounds by simulating natural photosynthesis. Here, inspired by organic photovoltaic structures, we demonstrate a bio-artificial photosynthetic system based on the hybridization of polymer semiconductor films and bacteria. The study suggests that the polymer-based semiconductor film can efficiently drive the non-photosynthetic bacteria to convert CO₂ to acetate. By systematically characterizing the charge transport behavior of the bio-artificial photosynthetic system, the bulk-heterojunction structure and charge transport layers are proven to enhance the system performance markedly. The scalable floating artificial bio-leaf system can produce acetate to gram scale in a week. Notably, the semiconductor film is easy to recycle and maintains stable performance, showing good sustainable production capability of the system. A quasi–solid-state artificial bio-leaf is successfully prepared using agar to simulate the morphology and function of natural leaves. Last, the acetate production converted from CO₂ was used to grow yeast for food production, thus achieving a complete simulation of natural photosynthesis.

Artificial bio-leaves constructed with polymer semiconductor films and bacteria are demonstrated to convert CO₂ to acetate.

INTRODUCTION

Photosynthesis is an essential activity on Earth that can fix CO₂ into sugars or other organic matter. By learning from natural photosynthesis, biohybrid artificial photosynthesis can also convert CO₂ and water to multicarbon compounds and gradually emerge as a promising sustainable technique to transform solar energy into chemical energy (1, 2). The biohybrid artificial photosynthetic system combines the robust light-harvesting abilities of semiconductors with the highly effective catalytic functions of biological cells and enzymes, providing great potential for biosynthesis (3). Because silicon nanowires were initially introduced into biohybrid artificial photosynthetic system (4), various inorganic nanoscale semiconductor and organic conjugated semiconductor materials have been developed, such as cadmium sulfide nanoparticles (5), gold nanoclusters (6), copper zinc tin sulfide nanocrystals (7), indium phosphide quantum dots (8), organic perylene diimide derivative, poly(fluorene-co-phenylene) (9), and organic dyes (10–12). The semiconductor materials adhere to the surface or enter the interior of bacterial cells, effectively transferring energy to the bacteria and enhancing the sunlight utilization efficiency.

Different functional artificial photosynthetic systems have been studied and applied in fields such as CO₂ reduction, water oxidation, and nitrogen reduction to NH₃ (5, 6, 13–15). These successful attempts demonstrate the enormous advantages of combining semiconductors and bacteria cells for artificial photosynthesis. However, the current bio-artificial photosynthetic systems still require solving some thorny issues, such as the nanoscale semiconductor materials and organic small molecules hardly being recycled from the system. As bacterial cells age, the semiconductor materials can no longer be collected or extracted from the bacteria’s surface or interior. Current bio-artificial photosynthetic systems also usually suffer from low sunlight utilization efficiency (5, 15–17). Among the key reasons is that the reported semiconductor materials are single components lacking efficient excitons separation interface, leading the systems to exhibit severe charge recombination. In addition, some bio-artificial photosynthetic systems still need the external wire to connect the photoanode and the biological electron acceptor (2, 12, 13, 18, 19). Additional external connections not only increase manufacturing costs but also are inconvenient to use. Constructing a natural leaf-like sustainable bio-artificial photosynthetic system with a simple wireless structure and recycled semiconductor capability is still challenging.

With strong light-absorption capabilities, accessible processing properties, and high exciton separation efficiency, conjugated polymer semiconductors are widely adopted to develop thin-film solar cells (20–23). Inspired by the outstanding performance of polymer semiconductor films in organic photovoltaics (OPVs), we propose introducing polymer semiconductor films to construct a recyclable and highly efficient bio-artificial photosynthetic system. The polymer semiconductor film was deposited on the indium tin oxide (ITO) or glass substrate, serving as the light-harvesting layer and providing a large-area contact interface with bacteria. The non-photosynthetic bacteria were cultured on the semiconductor film to form the CO₂ reduction bio-artificial photosynthetic system. The polymer semiconductor film strongly absorbs the light and efficiently generates the excitons. The electrons can be efficiently separated and transferred to the bacteria to drive CO₂ reduction to acetate. Compared with traditional artificial photosynthetic systems constructed with inorganic nanoparticles, the polymer film–based bio-artificial photosynthetic system exhibits robust charge generation and separation performance, resulting in the light-utilization quantum efficiency (QE) of up to 11%, which is among the highest efficiency of the reported systems in the field.

A scalable floating bio-artificial photosynthetic system was constructed by adopting the flexible substrate, and the freestanding quasi–solid-state bio-artificial leaves were also prepared to demonstrate a simulation of the natural plant leaves. This artificial leaf system, composed of bacteria and organic semiconductor films, boasts a simple structure and is easily recyclable and scale-up expandable. The produced acetate was then used to grow yeast for food production (24). The entire process completes the simulation of the natural photosynthesis process from CO₂ to food. This study on bio-artificial leaves may inspire further research on organic semiconductor materials and engineered bacteria to explore more applications and enhance the conversion efficiency of this bio-artificial photosynthetic system.

RESULTS

Bio-artificial photosynthetic system hybrid with polymer semiconductor film and S. ovata bacteria

Poly(3-hexylthiophene) (P3HT) is a well-known polymer semiconductor broadly used in OPV field (fig. S1) (25, 26). The outstanding photovoltaic performance, good chemical stability in aqueous solution, and superior biocompatibility shown in previous studies indicate good potential to be adopted in artificial photosynthetic systems (25). Here, a P3HT film was prepared on the substrate by the spin-coating method for the large-area light-harvesting layer. Subsequently, the P3HT film was placed in the medium solutions that contained non-photosynthetic Sporomusa ovata (American Type Culture Collection, 35899) pre-cultured bacterial fluids for bacterial film culture. After around 96 hours of the cell culture process, the optical density at 600 nm (OD₆₀₀) of S. ovata bacteria solution reached 0.3 to 0.4, indicating that the bacteria were ready to start the subsequent artificial photosynthetic experiments.

Figure 1 illustrates the electron transfer principle of the bio-artificial photosynthetic system. As the light-absorbing layer, P3HT film produces excitons upon being excited by sunlight. A part of the photogenerated electrons directly reaches the active site of the bacterial membrane in contact with the organic semiconductor film. The other part is transferred indirectly to the active site of bacteria via an electron mediator {potassium ferricyanide [K₃Fe(CN)₆]} (8). In addition, the added hole-trapping agent cysteine captures holes to produce cystine and H⁺ (5, 8, 27). The H⁺ then participates in the Wood-Ljungdahl pathway (WLP) metabolic process through the reaction of hydrogenases (fig. S2). The S. ovata bacteria then use the obtained energy to efficiently drive CO₂ reduction to acetate through the WLP (28).

Fig. 1. Schematic diagram of the electron transfer principle of the bio-artificial photosynthetic system.

Figure 2A shows a schematic diagram of the exciton generation, separation, and transportation process in the films with P3HT, [6,6]-phenyl-C₆₁-butyric acid methyl ester (P3HT:PCBM) heterojunction [bulk-heterojunction (BHJ)], and ITO/ZnO/P3HT:PCBM/MoO₃ multilayer film structures. The BHJ and multilayer film structure can suppress the exciton recombination and enhance the charge transfer efficiency. Inspired by previous studies, the polymer semiconductor–based bio-artificial photosynthetic system was constructed by co-incubating polymer semiconductor material films with bacteria (5, 6, 8). Compared with discrete bacteria-based artificial photosynthetic systems in previous reports, the polymer semiconductor film–based artificial photosynthetic system easily monitors the state of semiconductor materials. In addition, this planar-structured polymer semiconductor film is more easily recycled than the nanoscale size of semiconductor materials from the systems. The scanning electron microscopy (SEM) images show that all the substrates are smooth and clean, and the S. ovata bacteria can grow well and form a bacterial film on the substrates, indicating the good biocompatibility of the substrates. Notably, the S. ovata bacteria have a relatively dispersed bacterial distribution when the hybrid systems are constructed with bare glass or ITO substrates (fig. S3). We infer that, due to the lack of photogenerated electron supply from semiconductor films, the bacteria growing in the bare glass and ITO-based systems can only grow by their metabolic pathways, and the growth and division rates are slower than in the systems containing polymer semiconductor films. Therefore, the density of the bacteria films on the bare glass and ITO substrates is relatively sparse. The SEM image in Fig. 2B shows the morphology change of the polymer semiconductor films before and after culturing the bacteria cells. After bacteria culturing, the S. ovata bacteria grew densely, forming a bacterial film covered on the polymer semiconductor–based substrate. The viability of S. ovata in the bio-artificial photosynthetic system was evaluated by assessment of its OD₆₀₀ (Fig. 2C). The S. ovata cell with P3HT film demonstrated superior growth under simulated light conditions, again suggesting the excellent biocompatibility of polymer-based semiconductor films.

Fig. 2. Photosynthesis test of polymer semiconductor film–based biohybrid artificial photosynthetic systems.

(A) Schematic diagrams of exciton separation and recombination of polymer semiconductor films with P3HT, P3HT:PCBM bulk-heterojunction (BHJ), and multilayer film structures. CB, conduction band; VB, valence band. (B) Scanning electron microscopy (SEM) characterization of the S. ovata@Glass/P3HT and S. ovata@ITO/ZnO/P3HT:PCBM/MoO₃ artificial biohybrid system. The scale bar is 5 μm. (C) The OD₆₀₀ of S. ovata in different polymer semiconductor film–based systems during artificial photosynthesis test. (D) The total acetate production and (E) the cumulative variation of acetate production during a week of testing artificial photosynthesis under various conditions. (F) The QE of S. ovata hybrid artificial photosynthetic systems with different structured organic semiconductor films.

The photosynthesis performance of the prepared system was then tested under light irradiation conditions over 1 week (Fig. 2, D and E, and fig. S4). The results indicate that the P3HT film enhanced the acetate yield of the system under the same test conditions, demonstrating the promising capability to convert CO₂ to acetate of the hybrid bio-artificial photosynthetic system. Following previous investigations, the QE of the artificial photosynthetic process can be calculated by the ratio of the total acetate production to the total number of photons (6). The QE of the S. ovata@P3HT/bio-artificial photosynthetic system reaches about 2.5%, which is obviously higher than 0.5% of the control system without polymer semiconductor film (Fig. 2F). The P3HT film produces photogenerated electrons that act as a photosensitizer under light conditions. A large number of photogenerated electrons are then transferred directly to bacteria through the polymer semiconductor film/bacteria interface or transferred indirectly through an electron mediator to promote artificial photosynthesis performance and enhance acetate production yield (8). We found that the transparent ITO electrode also benefits the acetate yield in the polymer semiconductor film–based hybrid artificial photosynthetic systems, indicating that the transparent electrode can also contribute to the charge utilization efficiency of the systems. We infer that ITO can efficiently collect electrons from polymer semiconductor films and further transfer electrons to electron mediators, thereby promoting the efficiency of the system in converting CO₂ into acetate compared with the system constructed with glass substrate. The results from preliminary research demonstrate the promising performance when applying polymer semiconductor films in bio-artificial photosynthetic systems.

Construction of bio-artificial photosynthetic systems with multilayer semiconductor films and S. ovata bacteria

Inspired by OPV device structures, the BHJ structures consisting of donor and acceptor are expected to improve the excitons separation efficiency. Because fullerenes are one of the famous donors and P3HT:PCBM BHJ has been broadly researched in the OPV field, we adopted P3HT:PCBM organic semiconductor film as the light-harvesting layer in our constructed bio-artificial photosynthetic system. In addition, the charge-selective layers, including the electron transport layer (ETL) and hole transport layer (HTL), can also suppress charge recombination by efficiently extracting electrons and holes from the active layer in OPVs (29). To further enhance the exciton separation efficiency and charge transport capability, the energy level–matched ZnO ETL and MoO₃ HTL layers were incorporated on both sides of the active film to form a multilayer-structure bio-artificial photosynthetic system. Figure S5 illustrates the energy level diagram of the ITO/ZnO/P3HT:PCBM/MoO₃ structure (18, 30). A reasonable energy level arrangement ensures efficient energy transfer from the active layer of the organic semiconductor to the active bacterial site.

The charge generation process of the bio-artificial photosynthetic system can refer to OPVs. The excitons generated in P3HT:PCBM layer under light irradiation, then separated at the BHJ interface and transferred to the charge transport layer, and lastly arrived at the electrode and interface of the aqueous solution. Anions in solution capture a portion of the holes, and another portion is quenched by the hole-trapping agent cysteine. The electrons transfer to the active site of S. ovata by the electron mediator and then participate in the internal metabolic processes of the bacteria. Figure S6 illustrates the transport of photogenerated electrons to the S. ovata cell membrane with the assistance of the electron mediator, resulting in an increased production of electron-rich H₂ from H⁺ by hydrogenase (2, 6, 8, 31). CO₂ can be efficiently reduced to acetate through its metabolic pathway. Although the electron mediator can effectively enhance the electron transfer efficiency from semiconductors and bacteria, its susceptibility to environmental loss not only limits the sustainability of the system but also leads to higher system costs and operational complexity. Developing electron media–independent artificial photosynthetic systems will be an essential research topic in future research of artificial photosynthetic systems. On the one hand, exploring environmentally friendly electron mediators that are easy to recycle to replace potassium ferrocyanide is essential; on the other hand, constructing artificial photosynthetic systems that do not require electron mediators is also a critical approach to solving the above problems (32, 33).

The P3HT:PCBM organic semiconductor film and S. ovata hybrid artificial photosynthetic systems were constructed and optimized in detail, including film preparation, bacterial culture, lighting conditions, and film area (figs. S7 to S17). The artificial photosynthetic systems were constructed with 10 ml of S. ovata medium and 2 cm–by–2 cm substrate with polymer semiconductor film in 40-ml anaerobic vials. The vials were operated in a shaker at 34°C under an N₂:CO₂ (80:20) gas atmosphere with 10 hours of light and 14 hours of darkness per day. A white light-emitting diode (LED) with a light intensity of 10 mW/cm² was used as the light source. The concentration of electron mediator was optimized to 1 mM by a set of experiments including the bacteria growth and acetate yield tests, which is shown in fig. S12. The OD₆₀₀ of S. ovata revealed that both ITO/P3HT:PCBM and ITO/ZnO/P3HT:PCBM/MoO₃ films promote the growth of S. ovata bacteria (Fig. 2C). The S. ovata proliferated faster in the multilayer film artificial leaf system, indicating that the multilayer film bio-artificial photosynthetic system has more photogenerated electrons transferred to S. ovata to promote its growth. Further studies suggest that the sufficient electrons and an efficient electron transfer process play the important roles in improving bacteria growth (fig. S15).

The photosynthesis performance of the hybrid bio-artificial photosynthetic system was systematically studied. Figure 2 (D and E) and fig. S4 show the cumulative changes in acetate yield over 1 week. The total acetate yield of the system with BHJ film (S. ovata@ITO/P3HT:PCBM) is higher than that of the system with P3HT layer (S. ovata@ITO/P3HT). Consistent with the active layer in OPVs, the BHJ blend film can increase the effective interface area and form interpenetrating continuous charge transport channels and ultimately reduce the exciton recombination rate and enhance the excitons separation efficiency (34). As a result, the acetate production yield is markedly increased. The multilayer-based system (S. ovata@ITO/ZnO/P3HT:PCBM/MoO₃) obtained the best photosynthesis performance with an acetate yield of up to 83 mg, which is almost double the production of 42 mg for the BHJ film system. The results indicate that the organic semiconductor film efficiently accomplishes the separation and transport of electrons and holes through ETL, HTL, and ITO electrode. The sufficient electron supply markedly enhances bacterial metabolism, efficiently reduces CO₂, and greatly improves acetate production. We also conducted control experiments constructed by dead S. ovata bacteria cells (OD₆₀₀ of 0.57) and polymer semiconductor films to confirm that acetate was produced by living S. ovata bacteria. The results clearly show that the OD₆₀₀ of the control groups constructed by dead bacteria remained unchanged, and no acetate production was detected (fig. S18), indicating that CO₂ cannot converted to acetate by dead bacteria, and the well-living bacteria are essential for the artificial photosynthesis process. Figure 2F demonstrates that the QE of the system with BHJ film was ~4 to 5%, while the multilayer-based system exhibited a QE in the range of 10 to 11%, which is among the highest QE results in reported bio-artificial photosynthetic systems (table S1).

Characterization of bio-artificial photosynthetic systems

To investigate the electron transport process between the organic semiconductor film and S. ovata bacteria in the hybrid bio-artificial photosynthetic system, the morphology and photoelectric response behavior of the system were characterized and investigated. On the multilayer films, S. ovata can also form a dense bacterial film (Fig. 2B). The S. ovata still presents a good shape, indicating again excellent biocompatibility of the organic semiconductor film. Figure S19A depicts the correlation between the deposition ratio of S. ovata and their growth state. The deposition ratio decreased with the increased OD₆₀₀ of S. ovata, which suggests that lots of free bacteria remain in the culture medium after the bacteria form a bacterial film, which also contributes to the yield of photosynthesis products.

The absorption spectra of various organic semiconductor films are presented in fig. S19B. Because P3HT film strongly absorbs the blue and green light, the active layers present a red-brown color. According to the external QE results shown in fig. S19C, the BHJ film with the P3HT:PCBM blend is more beneficial to the photovoltaic conversion process than the individual components. On the basis of the results of optoelectronic response measurement, the P3HT:PCBM heterojunction film displayed higher photocurrent than the film with P3HT or PCBM individual components (Fig. 3A) (9, 35), and the photocurrent further increased with the integration of both ETL and HTL. As shown in fig. S19D, the photocurrent signal was changed from fast response to being smooth, implying that both the BHJ structure and the involvement of the transport layers enhance the efficiency of hole/electron separation.

Fig. 3. Characterization of S. ovata@ITO/ZnO/P3HT:PCBM/MoO₃ bio-artificial photosynthetic system.

(A) Representative photocurrent responses of ITO, ITO/P3HT, ITO/PCBM, ITO/P3HT:PCBM, and ITO/ZnO/P3HT:PCBM/MoO₃ polymer semiconductor films at 10-s intervals, which were recorded in phosphate buffer (0.1 M, pH 7.4) containing 0.2 wt % cysteine at 0-V bias. (B) Photocurrent response of S. ovata, ITO/ZnO/P3HT:PCBM/MoO₃, and S. ovata@ITO/ZnO/P3HT:PCBM/MoO₃ artificial biohybrid system in 10 mM PBS solution (pH 7.4) at 0-V bias. (C) Schematic diagram of the arrangement for measuring the ionic voltage (top) and device voltage (bottom) from polymer semiconductor film. (D) The ionic voltage and device voltage changes in response to light activation in ITO/ZnO/P3HT:PCBM/MoO₃ film recorded using a free-floating electrode tip in phosphate-buffered saline bath solution (0.1 M, pH 7.4).

To further understand the process of photoexcited electron transfer, we tested the photoelectric response behavior of the multilayer-based bio-artificial photosynthetic system by a potentiostatic module combined with a platinum electrode and a saturated calomel electrode (SCE) (9, 36). The working electrode was directly connected to the ITO layer with a metal clip in the system. The multilayer film area of ∼1 cm² was illuminated (Fig. 3B and fig. S19E) (36). The S. ovata bacterial film does not respond to light irradiation. With the strong photovoltaic effect, the ITO/ZnO/P3HT:PCBM/MoO₃ layers show a robust response to light. The measured photocurrent achieves to −0.32 μA/cm². After cultured with the bacterial film, the photogenerated electrons are efficiently transferred to the S. ovata in the solution of bio-artificial photosynthetic system, resulting in a notable reduction in the photocurrent to only ~−0.09 μA/cm² measured from S. ovata bacterial film in the multilayer-based system.

Patch clamp is broadly used to measure the photovoltage and ionic voltage of a device to characterize the interaction of the device and biological interfaces (37). The photovoltage alterations were also explored by measuring the voltage relative to bath grounding and analyzing their impact on the ionic charge within the electrolyte solution (Fig. 3C, top image). The ionic voltage of the film quickly shifts a peak at the instant the irradiation initiates and then decays back to baseline within 1 to 3 ms (Fig. 3D and fig. S19F). This observation suggests that the anions in the solution respond quickly to changes in holes. The positive terminal of the patch-clamp amplifier was connected directly to the ITO layer of the film to measure the intrinsic photovoltage response, as shown in the bottom image of Fig. 3C. In response to 5 s of light stimulation, the film underwent a quick initial alteration in photoinduced voltage, and, eventually, the change leveled off and stabilized at a negative value under light irradiation. The photovoltage and ion voltage of the film were observed to vary in opposite directions, indicating that electrons and holes can be effectively separated by multilayer film structures (37).

The scalable floated bio-artificial photosynthetic system

The long-term stability of the multilayer-based bio-artificial photosynthetic system was then evaluated. During a 2-week artificial photosynthesis test, the absorption range and intensity of the semiconductor film remained constant (Fig. 4A), and the bio-artificial photosynthetic system consistently produced stable yields of acetate (fig. S20). The excellent stability and biocompatibility, combined with the lightweight and flexible properties of the organic semiconductor films, make the bio-artificial photosynthetic system an excellent candidate for simulating natural floating leaves. The organic semiconductor film prepared in the shape of a lotus leaf on the flexible polyethylene naphthalate (PEN) substrate was used to simulate the state of a natural aquatic leaf (Fig. 4B, inset, top image). By adjusting the hydrophilicity of the organic semiconductor film, the artificial leaf can float on the surface of the water or sink to the bottom (Fig. 4B, inset, bottom image). The artificial photosynthesis performance of the artificial leaves was subsequently tested. The OD₆₀₀ of S. ovata is close in both floating and submerging artificial leaf systems (fig. S21A). The total acetate production of the floating leaf was comparable with the yield in the traditional submerged state (Fig. 4B and fig. S21, B and C). The results suggest that the floating scheme is also a promising approach for artificial leaves with little effect on artificial photosynthesis.

Fig. 4. Performance of a floating, scalable, and recyclable artificial bio-leaf system for artificial photosynthesis.

(A) Absorption spectra of ITO/ZnO/P3HT:PCBM/MoO₃ polymer semiconductor film before and after a 2-week period of artificial photosynthesis testing; the inset is a comparison picture. (B) The total acetate yield of the ITO/ZnO/P3HT:PCBM/MoO₃ polymer semiconductor film in the artificial biohybrid system while immersed in the medium and the floating state. (Inset: Multilayer film 1: simulating the shape of a natural leaf; and multilayer film 2: the film shape used in this work. Pictures of the artificial biohybrid system in submerged and floating states.) (C) The total acetate yield of the artificial leaf system following scaling up of the multilayer film area. The demonstration of a large floating leaf in a lotus pond (inset). (D) OD₆₀₀ and acetate yield in artificial photosynthesis test with multilayer film cycling for three cycles of an artificial biohybrid system. (E) Photograph of quasi–solid-state artificial leaf. (F) Comparison of the acetate yield for artificial photosynthesis test using a quasi–solid-state artificial leaf and a solution state artificial photosynthetic system.

To develop the artificial leaf system further, we scaled up the size of the artificial leaf to increase the production quantity and explore the application field. The optimal artificial leaf area was determined in different volumes of the artificial leaf system solutions (fig. S21D). The acetate yields of scaled-up systems increase linearly with the artificial leaf area (fig. S21, E and F), which suggests that the system has considerable feasibility of scaling up. The prepared large-area flexible organic semiconductor films were placed in the natural environment of the pond to demonstrate the state of the artificial leaf (inset of Fig. 4C). The acetate yield of 8.2 g/week was obtained from an artificial leaf system with a 400-cm² area of organic semiconductor films (Fig. 4C). Gram-scale production demonstrates excellent potential for further large-scale industrial production applications.

Notably, the multilayer semiconductor films on the substrate can be easily recycled from the bio-artificial photosynthetic system, which provides a feasible way to recycle semiconductor materials when bacterial growth is not good. After a 1-week artificial photosynthesis process, the multilayer semiconductor films on the substrate were recycled and cleaned. Subsequently, the recycled multilayer films were placed in a fresh bacterial solution to take the next cycle of artificial photosynthesis process. During the three-cycle tests, the bio-artificial photosynthetic system maintains stable acetate production, and the total acetate yields are almost unchanged (Fig. 4D). The superior long-term stability and recyclability indicate good sustainability in CO₂ reduction of the bio-artificial photosynthetic system.

Except for aquatic plants, most natural leaves of land plants perform photosynthesis in the form of solid-state leaves in the air environment. To simulate the natural photosynthesis process of natural leaves, we prepared a quasi–solid-state artificial leaf and conducted photosynthesis experiments. The medium was prepared in the shape of a leaf with agar. The bacteria film was then cultured on the organic semiconductor films and assembled with medium agar to form a quasi–solid-state artificial bio-leaf (Fig. 4, E and F). With 24 hours of light irradiation, the quasi–solid-state artificial leaf successfully produced the acetate in the CO₂/N₂ atmosphere. The acetate yield of the artificial bio-leaf is about one-fourth of the yield for the system in the solution medium. Within the first 72 hours, the yield of acetate production continues to increase and later remains unchanged. The quasi–solid-state artificial bio-leaf system presents a sunlight utilization efficiency of up to 4% during the first 72 hours of the artificial photosynthesis process and remains constant later (fig. S22). The lower sunlight utilization efficiency of artificial bio-leaves was compared with that of the solution-based systems. We infer that the hydrogel system is not favorable for the dissolution of CO₂ and transfer in the quasi–solid-state medium, which limits the acetate yield of the artificial bio-leaf. Although the photosynthesis performance of artificial bio-leaf is still low, the artificial photosynthesis process of the quasi–solid-state artificial bio-leaf does not need to be carried out in the aqueous solution. The constructed artificial bio-leaf can vividly simulate the state of the natural photosynthesis process, which is more valuable as a reference for developing simple and efficient artificial bio-leaves.

The acetate produced by the artificial leaf system further converted into food

Some previous creative research has successfully demonstrated that food and other organic products can be produced through acetate converted from carbon dioxide, allowing food production independent of biological photosynthesis (24). Acetate can serve as both a carbon source and energy source for yeast, and combining this carbon fixation with an artificial leaf system may provide an effective strategy for food production (Fig. 5A). We separated the S. ovata bacteria from the medium solution of the artificial leaf system by centrifugation and then used the supernatant to culture yeast. Yeast can be successfully inoculated and multiplied in the medium solution containing artificial leaf-produced acetate. The OD₆₀₀ of yeast increased ~5.5-fold, and the dry weight increased ~5.6-fold compared to growth in S. ovata medium without organic semiconductor films (Fig. 5, B and C). Although the yeast grows only half as fast as in the medium containing glucose, the whole process is still encouraging because the food can be produced from CO₂ with sunlight as the only energy source by the artificial leaf system.

Fig. 5. The produced acetate is further used to grow yeast for food production.

(A) The artificial leaf system reduces CO₂ to acetate to support the growth of food-producing organisms (yeast). Saccharomyces cerevisiae was grown at 30°C, shaking, in medium containing the product of the S. ovata, the S. ovata@ITO/ZnO/P3HT:PCBM/MoO₃ artificial system, and yeast malt medium with glucose, respectively. (B) OD₆₀₀ of yeast grow in different medium over 96 hours. (C) The yeast dry weight after 96 hours of growth in different media.

DISCUSSION

The bio-artificial photosynthetic system is a promising platform for producing valuable products by converting CO₂, water, and other simple atmospheric substances. Semiconductor materials are one of the critical components in the system. To date, most semiconductors are adopted with the morphology of nanoparticles, nanocrystals, nanoclusters, or organic small molecules, which are generally challenging to recycle. In this work, we developed a bio-artificial photosynthetic system based on the polymer semiconductor film with S. ovata bacteria for CO₂ reduction. The semiconductor films with bulk heterojunction and multilayer structure exhibited excellent biocompatibility and enhanced electron/hole separation efficiency, as well as good stability and recyclability. Under optimized conditions, the bio-artificial photosynthetic system can efficiently convert CO₂ to acetate, and the sunlight utilization efficiency of the system can reach up to 11%, which is among the highest efficiency of reported artificial photosynthetic systems so far. The lightweight and flexible properties of the polymer-based semiconductor films enable the preparation of scalable floating artificial bio-leaf systems to simulate the natural photosynthesis of aquatic plants. Notably, a quasi–solid-state artificial bio-leaf was further demonstrated to simulate the land plant leaves. Last, the produced acetate was used to grow yeast for food production and complete the process of converting CO₂ into food. This work successfully attempted to simulate natural plant leaves by introducing polymer semiconductor film, providing an effective solution for designing a bio-artificial photosynthetic system for efficient solar-to-chemical energy conversion.

MATERIALS AND METHODS

Organic semiconductor film preparation

P3HT regioregular (RIEKE) and PCBM (Solarmer) were purchased and used without any further purification. ZnO nanoparticles were synthesized in the laboratory based on prior literature (38). The ITO substrate was cleaned several times with deionized water and isopropanol in an ultrasonic bath. Plasma cleaning completed the surface treatment process of the substrate. The ZnO nanoparticles were spin coated on the ITO substrate at 4000 rpm. P3HT:PCBM solution of 20:20 mg/ml was prepared, and, then, the solution was heated at 50°C with stirring and then deposited on the ITO/ZnO substrate by spin coating at 1000 rpm for 1 min. The obtained organic layer was then annealed at 120°C for 10 min. Last, a complete ITO/ZnO/P3HT:PCBM/MoO₃ organic semiconductor film was obtained by depositing a 10 nm MoO₃ layer on the top by thermal evaporation. The floating polymer semiconductor films with the same structure were obtained by replacing the glass substrate with a flexible PEN substrate.

Construction of an organic semiconductor film and S. ovata hybrid bio-artificial photosynthetic system

The organic semiconductor film was irradiated under UV light over 1 hour for sterilization. It was then placed into the S. ovata culture solution already grown in DSMZ-311 medium for 48 hours and continued to be incubated for 48 hours at 34°C under N₂/CO₂/H₂ (80/10/10) atmosphere. After centrifugation at 6000 rpm for 10 min, the pellet was resuspended in 10 ml of M9-MOPS: Morpholinepropanesulfonic acid medium perform artificial photosynthesis tests at 34°C under N₂/CO₂ (80/20) atmosphere. The OD₆₀₀ and acetate yield were measured every 24 hours during the whole process.

Artificial photosynthesis test

Simulated sunlight for 12 hours and darkness for 12 hours at 34°C under N₂/CO₂ (80/20) atmosphere was set as the photosynthesis condition. The LED full-spectrum white light is used as an irradiation source with adjustable light intensity. Then, cysteine and potassium ferricyanide were added every 24 hours as hole-trapping agents and electron mediators, respectively. The mixture was centrifuged at 6000 rpm for 10 min every 24 hours, and the acetate concentration of the supernatant was measured while the pellet continued to be resuspended in M9-MOPS medium for the artificial photosynthesis process. The test conditions were the same with various groups. Three parallel groups for each experiment were performed to assess the reliability.

Construction of a floating bio-artificial photosynthetic system

The organic semiconductor film prepared on a PEN flexible substrate was cut out in a suitable leaf shape. A layer of polyurethane foam was uniformly sprayed on the backside of the PEN substrate, dried naturally and then placed in S. ovata medium already pre-cultivated for 48 hours, and continued to be cultured for 48 hours at 34°C under N₂/CO₂/H₂ (80/10/10) atmosphere.

Constructing quasi–solid-state artificial bio-leaf

The free-standing organic semiconductor films were prepared by taking 20 μl of chlorobenzene solution of P3HT:PCBM organic semiconductor and dropping it on a certain area of water surface prepared in advance and waiting about 2 min. After that, it was transferred to the agar medium already formed with the bacterial film to obtain the quasi–solid-state artificial bio-leaf. The quasi–solid-state artificial bio-leaf test system was conducted by replacing the liquid medium with agar medium at the beginning of the test in the conventional artificial photosynthetic system.

Photosynthetic product measurement

¹H nuclear magnetic resonance (NMR) was used to quantify the production of acetate (fig. S23). The sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate (TMSP-d4; Cambridge Isotope Laboratories Inc.) was the internal standard. Carbon source verification experiments confirmed that part of the acetate converts from CO₂ (figs. S24 to S26).

QE calculation

QE was defined by the ratio of the electrons consumed to produce acetate to the total input photons. It took eight electrons to reduce two CO₂ molecules to one acetate molecule. The following equation can be used to calculate the QE

$$\text{QE}\%=\text{electrons}/\text{photons}\times 100$$

$$=(8\times \text{mol acetate generated})/(\text{mol total photons})\times 100$$

$$=(8\times C\times V\times N_{\mathrm{A}})/({\mathrm{\phi }}_{\text{ph}}\times t\times A)\times 100$$

where C is the total acetate concentration (millimolar), V is the total suspension volume (milliliters), N_A is the Avogadro’s number of 6.022 × 10²³, ϕ_ph is the photo flux (per square centimeter per second), t is the reaction time (seconds), and A is the area of irradiation (square centimeters).

Photocurrent measurement

Photocurrent testing was carried out using a three-electrode system using an ITO electrode of organic semiconductor film as the working electrode, a SCE as the reference electrode, and platinum as the counter electrode in phosphate-buffered saline (PBS; pH 7.4) containing cysteine. The light source was a full-spectrum white LED panel, and a frequency generator was used to control the light duration and intensity. All measurements were performed at room temperature with no bias voltage applied.

Measurement of device and ionic photovoltages

The device and ionic photovoltages of organic semiconductor film were measured by using a patch clamp (Taimen, Chengdu) in the current clamp mode. For the ionic photovoltage, the Ag/AgCl electrode was directly contacted with the MoO₃ layer without using a glass tube electrode. For the device photovoltage, the ITO layer was contacted with the Ag/AgCl electrode of the patch clamp. The grounded Ag/AgCl electrode remained in the solution (PBS, pH 7.4) during the test. The light source was a 520-nm green laser.

Supplementary Materials

This PDF file includes:

Supplementary Text

Figs. S1 to S26

Table S1

References