Light-driven, bias-free nitrogenase-based bioelectrochemical cell for ammonia generation.

Nitrogen fixation is a key process that sustains life on Earth. Nitrogenase is the sole enzyme capable of fixing nitrogen under ambient conditions. Extensive research efforts have been dedicated to elucidating the enzyme mechanism and its artificial activation through high applied voltage, photochemistry, or strong reducing agents. Harnessing light irradiation to minimize the required external bias can lower the process's high energy investment. Herein, we present the development of photo-bioelectrochemical cells (PBECs) utilizing BiVO4/CoP or CdS/NiO photoanodes for nitrogenase activation toward N2 fixation. The constructed PBEC based on BiVO4/CoP photoanode requires minimal external bias (200 mV) and suppresses O2 generation that allows efficient activation of the nitrogenase enzyme, using glucose as an electron donor. In a second developed PBEC configuration, CdS/NiO photoanode was used, enabling bias-free activation of the nitrogenase-based cathode to produce 100 µM of ammonia at a faradaic efficiency (FE) of 12%. The ammonia production was determined by a commonly used fluorescence probe and further validated using 1H-NMR spectroscopy. The presented PBECs lay the foundation for biotic-abiotic systems to directly activate enzymes toward value-added chemicals by light-driven reactions.

Biotic-abiotic hybrids for bioanalytics and biocatalysis.

The advances in biotic-abiotic interfaced systems open new directions toward bioanalytics and biocatalysis applications. Conjugating the unique electronic and optic properties of nanoelements with the high selectivity and extraordinary catalytic abilities of biotic materials holds great promise to gain superior new features. Herein, we present a wide scope of biotic-abiotic research, with key examples for its utilization in bioanalytics applications as well as in biocatalysis. The described configurations feature methodologies that enable extending the known scientific toolbox to gain synergy. These new nanobiohybrids may contribute to major global challenges, for example, developing alternative energy utilization or new affordable biodiagnostics and therapeutics tools.

An Oxygen-Insensitive biosensor and a biofuel cell device based on FMN l-lactate dehydrogenase.

Lactate sensing has high importance for metabolic disease diagnostics, food spoilage, sports medicine, or the construction of biofuel cell devices. Therefore, continuous lactate sensing devices which enable accurate detection should be developed. Here we present the overexpression and utilization of FMN-lactate dehydrogenase from Saccharomyces cerevisiae for oxygen-insensitive, continuous amperometric lactate biosensing. The developed sensors exhibit a high signal-to-noise ratio, low interference effect, and a wide range of linear responses using both direct and mediated electron transfer configurations. The thionine-based mediated electron transfer configuration was stable for 8 h of continuous activity and two weeks of periodic activity with storage at 4 °C. We further grafted the redox mediators on multiwall carbon nanotubes to lower the redox mediator leaching effect. The developed grafting technique improved the biosensor stability and allowed continuous operation for at least 20 h. Both the mediator-entrapped and the grafted bioanodes were further coupled with a bilirubin oxidase-based biocathode to construct a biofuel cell device. The various biofuel cells have generated a maximal power output of 110 µW/cm2 under atmospheric conditions and 200 µW/cm2 under oxygen saturation.

Production of photocurrent and hydrogen gas from intact plant leaves.

Here, we show that it is possible to harvest photocurrent directly from unprocessed plant tissues from terrestrial or aquatic environments in bio-photoelectrochemical cells (BPECs) and use the current to produce molecular H2. The source of electrons is shown to originate from the Photosystem II water-oxidation reaction and utilizes exported mediating molecules, especially NADPH. The photocurrent production is dependent on the concentration of the photosynthetic complexes, as an increase in total chlorophyll and oxygen evolution rates in the leaves lead to increased photocurrent rates. The permeability of the outer leaf surface is another important factor in photocurrent harvesting. Different tissues produce photocurrent densities in the range of ∼1-10 mA/cm2 which is significantly higher than microorganism-based BPECs. The relatively high photocurrent and the simplicity of the plants BPEC may pave the way toward the development of future applicative photosynthetic based energy technologies.

First-Principle Colloidal Gate for Controlling Liquid and Molecule Flow Using 2D Claylike Nanoparticles.

Herein, we exploit the natural tendency of two-dimensional (2D) clay nanoparticles to self-assemble and restrict water permeability in soils to fabricate a first of its kind synthetic, pH-activated, reversible, and tunable colloidal flow gate. To realize this, we studied the effect of the pH level of a suspension of claylike layered double hydroxide (LDH) nanoparticles on the LDH coagulation process. We then packed the LDH into a fixed-bed column and examined the effect of pH on mass transport through the column. We found that the 2D platelike LDH particles coagulate in an edge-to-edge configuration, which renders highly nonisotropic aggregates, pivotal for obstructing the transport of liquid and molecules therein. We showed that the coagulation and flow through the column may be regulated by imposing various pH levels as an external stimulus to affect LDH zeta potential. Hence, this work shows that the flow through a column comprising a 2D particle bed can be regulated in a reversible manner by simply alternating the pH of the wash solution, equilibration time, or gate dimensions. Furthermore, we show that, subject to pH treatment, we may open and close the colloidal gate for the transport of large molecules and provide selective transport thereof.

The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer.

Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme's internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes.

Protein-Mediated Biosynthesis of Semiconductor Nanocrystals for Photocatalytic NAD(P)H Regeneration and Chiral Amine Production.

The use of predesigned bioengineered proteins for self-grown nanomaterials is a promising strategy that opens new scientific directions for biotic-abiotic nano-bio hybrid configurations. The unique properties of nanomaterials can alter the original biological paradigm to allow novel metabolic routes or new activation triggers. In this work, we present a synthetic methodology for self-grown cadmium sulfide quantum dots in a 12-mer bioengineered stable protein 1 under ambient conditions. The sized controlled crystalline QDs are characterized and utilized for NADPH regeneration that is in turn used for the activation of the imine reductase enzyme. The presented nano-bio hybrid system enables the production of a single enantiomeric product that is required for the pharmaceutical industry. Our designed system presents superior activity and can continuously operate for at least 22 hrs with 82 % conversion efficiency. The obtained results may lay the foundations for future nano-bio hybrid systems that can operate both in vitro and in vivo.

Bioelectricity generation from live marine photosynthetic macroalgae.

The conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Bio-photoelectrochemical cells (BPECs) utilizing unicellular photosynthetic microorganisms have been studied, however similar harvesting of electrons from more evolved intact photosynthetic organisms has not been previously reported. In this study, we describe for the first time BPECs containing intact live marine macroalgae (seaweeds) in natural seawater or saline buffer. The BPECs produce electrical currents of >50 mA/cm2, from both light-dependent (photosynthesis) and light-independent processes. These values are significantly greater than the current densities that have been reported for single-cell microorganisms. The photocurrent is inhibited by the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating that the source of light-driven electrons is from photosynthetic water oxidation. The current is mediated to the external anode via NADPH and possibly other reduced molecules. We show that intact macroalgae cultures can be used in large-scale BPECs containing seawater, to produce bias-free photocurrents, paving the way for the future development of low-cost energy solar energy conversion technologies using BPECs.

Tailoring Quantum Dot Sizes for Optimal Photoinduced Catalytic Activation of Nitrogenase.

Many efforts have been directed towards elucidating the nitrogenase structure, its biocatalytic activity, and methods to artificially activate it by external stimuli. Here, we investigated how semiconductor nanoparticles (NPs) with sizes ranging between 2.3-3.5 nm form nano-biohybrids with the nitrogenase enzyme and enable its photoinduced biocatalytic activity. We examined two homogenously synthesized quantum dots (QDs), CdS, CdSe, and two nitrogenase variants, the wild-type and a cysteine-mutated. We show that the cysteine-mutated variant does not enhance the hydrogen generation amounts, as compared with the wild type. Nevertheless, we show that the 2.3 nm-sized CdSe NPs facilitate an eightfold increase compared with larger CdSe NPs. The obtained results were investigated using electrochemical techniques, transmission electron microscopy, and further confirmed by time-resolved spectroscopic measurements, which allow us to determine the electron tranfer rate constant (kET ) of the different configurations.

Utilization of FAD-Glucose Dehydrogenase from T. emersonii for Amperometric Biosensing and Biofuel Cell Devices.

Flavin-dependent glucose dehydrogenases (FAD-GDH) are oxygen-independent enzymes with high potential to be used as biocatalysts in glucose biosensing applications. Here, we present the construction of an amperometric biosensor and a biofuel cell device, which are based on a thermophilic variant of the enzyme originated from Talaromyces emersonii. The enzyme overexpression in Escherichia coli and its isolation and performance in terms of maximal bioelectrocatalytic currents were evaluated. We examined the biosensor's bioelectrocatalytic activity in 2,6-dichlorophenolindophenol-, thionine-, and dichloro-naphthoquinone-mediated electron transfer configurations or in a direct electron transfer one. We showed a negligible interference effect and good stability for at least 20 h for the dichloro-naphthoquinone configuration. The constructed biosensor was also tested in interstitial fluid-like solutions to show high bioelectrocatalytic current responses. The bioanode was coupled with a bilirubin oxidase-based biocathode to generate 270 µW/cm2 in a biofuel cell device.

Photosynthesis Z-Scheme biomimicry: Photosystem I/BiVO4 photo-bioelectrochemical cell for donor-free bias-free electrical power generation.

Photo-bioelectrochemical cells that are based on photosynthetic proteins are drawing increased attention for both fundamental and applied research. While novel photosynthetic based systems have been introduced, further optimization in terms of stability and efficiency is required. Photosystem I has been utilized extensively in bioelectronic devices, often in conjugation with viologen moieties which act as electron acceptors. It has been shown previously that a partial reduction of oxygen to H2O2 can facilitate damage to proteins hence, limits their long-term activation. Here, we show a newly developed bias-free, donor-free photo-bioelectrochemical system that mimics the natural photosynthetic Z-scheme. Polymethylene blue and polybutyl-viologen were tailored to fit the photosystem I donor and acceptor sides, respectively. Furthermore, we show that by coupling the developed biocathode with a BiVO4/CoP photoanode, a power output of 25 µW/cm2 can be achieved. We further show that our configuration can minimize the damaging effect of H2O2 by two different pathways, oxidation at the photoanode or reduction by the polymethylene blue layer at the biocathode.

In vivo and in vitro protein mediated synthesis of palladium nanoparticles for hydrogenation reactions.

We report the biosynthesis of size confined palladium nanoparticles (Pd-NPs). The 2-3 nm size Pd-NPs were grown in 12-mer protein stable protein 1 (SP1), which serves as a template for the NP formation. We further show that by controlling the protein expression levels in the cells we can alter the cells' catalytic activity. The in vivo grown Pd-NPs were utilized in a hydrogenation reaction, converting acetylene feedstock into ethylene and ethane. The presented concept can be further used for a wide range of applications by exploiting the synergetic effect of the biotic elements with the abiotic ones.

Bismuth Vanadate/Bilirubin Oxidase Photo(bio)electrochemical Cells for Unbiased, Light-Triggered Electrical Power Generation.

The construction of bias- and donor-free photobioelectrochemical cells for the generation of light-triggered electrical power is presented. The developed oxygen reduction biocathodes are based on bilirubin oxidase (BOD) that originates from Myrothecium verrucaria (MvBOD) and a thermophilic Bacillus pumilus (BpBOD). Methods to entrap the BOD with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) redox molecules in a polydopamine layer are presented. A pH-independent, positively charged pyrenebetaine linker was synthesized, utilized, and led to a threefold improvement to the bioelectrocatalytic current. Both the developed polydopamine/ABTS/MvBOD and the pyrenebetaine/BpBOD biocathodes were further coupled with BiVO4 /cobalt phosphate water-oxidation photoanodes to construct biotic/abiotic photobioelectrochemical cells, which generated power outputs of 0.74 and 0.85 mW cm-2 , respectively. The presented methods are versatile, show the strength of biotic/abiotic hybrids, and can be further used to couple different redox enzymes with electrodes.

Investigating Protein-Nanocrystal Interactions for Photodriven Activity.

We illustrate how intermolecular interactions facilitate ATP-free electron transfer between either native or engineered MoFe protein (MoFeP) from nitrogenase and a CdS nanorod (NR) by following the reduction of H+ to H2. First, by varying the charge on the surface of the NR, we show the role of electrostatic interactions on MoFeP binding to the particle surface and subsequent H+ reduction. Next, the role of strong, semicovalent thiol-CdS interactions was tested using free cysteines on the MoFeP. By blocking free cysteines, we show that the presence of free thiols on the protein has little to no influence on CdS binding and resultant photocatalytic activity. We next studied methods to covalently bind the protein to CdS by modifying the free cysteines with dibenzocyclooctyne (DBCO) and reacting the CdS NRs capped with a mixture of negatively charged thioglycolic acid and thiol-PEG3-azide ligands. As compared to that of the unmodified proteins, a 32.2 ± 1.5% and 61.7 ± 2.1% increase in H2 production was observed from MoFeP and C-MoFeP, respectively. At last, to test the effect of both charge and covalent tethering, positively charged cysteamine/azide CdS NRs were reacted with DBCO-modified C-MoFeP, which showed little improvement over native C-MoFeP alone under irradiation. These results show the importance of both electrostatic associations between the NR and protein and covalently tethering the protein to the semiconductor surface for enhanced electron transfer and photodriven activity.

Multicatalytic, Light-Driven Upgrading of Butanol to 2-Ethylhexenal and Hydrogen under Mild Aqueous Conditions.

Microbes produce low-molecular-weight alcohols from sugar, but these metabolites are difficult to separate from water and possess relatively low heating values. A combination of photo-, organo-, and enzyme catalysis is shown here to convert C4 butanol (BuOH) to C8 2-ethylhexenal (2-EH) using only solar energy to drive the process. First, alcohol dehydrogenase (ADH) catalyzed the oxidation of BuOH to butyraldehyde (BA), using NAD+ as a cofactor. To prevent back reaction, NAD+ was regenerated using a platinum-seeded cadmium sulfide (Pt@CdS) photocatalyst. An amine-based organocatalyst then upgraded BA to 2-EH under mild aqueous conditions rather than harsh basic conditions in order to preserve enzyme and photocatalyst stability. The process also simultaneously increased total BuOH conversion. Thus, three disparate types of catalysts synergistically generated C8 products from C4 alcohols under green chemistry conditions of neutral pH, low temperature, and pressure.

Light-Driven Catalytic Upgrading of Butanol in a Biohybrid Photoelectrochemical System.

This paper reports the design and preparation of a biohybrid photoelectrochemical cell (PEC) that can drive the tandem enzymatic oxidation and aldol condensation of n-butanol (BuOH) to C8 2-ethylhexenal (2-EH). In this work, BuOH was first oxidized to n-butyraldehyde (BA) by the alcohol oxidase enzyme (AOx), concurrently generating hydrogen peroxide (H2O2). To preserve enzyme activity and increase kinetics nearly 2-fold, the H2O2 was removed by oxidation at a bismuth vanadate (BiVO4) photoanode. Organocatalyzed aldol condensation of C4 BA to C8 2-EH improved the overall BuOH conversion to 6.2 ± 0.1% in a biased PEC after 16 h. A purely light-driven, unbiased PEC showed 3.1 ± 0.1% BuOH conversion, or ~50% of that obtained from the biased system. Replacing AOx with the enzyme alcohol dehydrogenase (ADH), which requires the diffusional nicotinamide adenine dinucleotide cofactor (NAD+/NADH), resulted in only 0.2% BuOH conversion due to NAD+ dimerization at the photoanode. Lastly, the application of more positive biases with the biohybrid AOx PEC led to measurable production of H2 at the cathode, but at the cost of lower BA and 2-EH yields due to both product overoxidation and decreased enzyme activity.

Semiconductor-Based, Solar-Driven Photochemical Cells for Fuel Generation from Carbon Dioxide in Aqueous Solutions.

There has been active interest to identify new methods to reduce CO2 into usable fuel sources. In this work, we demonstrate two types of photo-electrochemical cells (PECs) that photoreduce CO2 directly to formate in aqueous solutions both in the presence and absence of external bias or additional electron sources. The photocathodes were either a CuFeO2 /CuO electrode or a bilayer of CdTe on NiO, whereas the photoanode was a bilayer of NiOx on CdS. The PECs were characterized by using both electrochemistry and spectroscopy, and the products formed from CO2 reduction were characterized and quantified by using 1 H NMR spectroscopy and ESI-MS. In addition, an organohydride catalyst was tested in conjunction with the PECs, which not only showed a significant gain of 85 times in CO2 reduction (27 µm formate without the catalyst, 2.3 mm formate with it) compared to the NiO/CdTe photocathode system but could also generate methanol under an external bias (10 µm).

Enhanced Hydrogen Production from DNA-Assembled Z-Scheme TiO2-CdS Photocatalyst Systems.

A wide range of inorganic nanostructures have been used as photocatalysts for generating H2. To increase activity, Z-scheme photocatalytic systems have been implemented that use multiple types of photoactive materials and electron mediators. Optimal catalysis has previously been obtained by interfacing different materials through aggregation or epitaxial nucleation, all of which lowers the accessible active surface area. DNA has now been used as a structure-directing agent to organize TiO2 and CdS nanocrystals. A significant increase in H2 production compared to CdS or TiO2 alone was thus observed directly in solution with no sacrificial donors or applied bias. The inclusion of benzoquinone (BQ) equidistant between the TiO2 and CdS through DNA assembly further increased H2 production. While the use of a second quinone in conjunction with BQ showed no more improvement, its location within the Z-scheme was found to strongly influence catalysis.

Electrostatically assembled CdS-Co3 O4 nanostructures for photo-assisted water oxidation and photocatalytic reduction of dye molecules.

Electrostatic assembly is used here to couple CdS nanorods with Co3 O4 nanoparticles into photocatalytic systems that simultaneously oxidize water and mediate electron transfer. Layered films of CdS nanorods and Co3 O4 nanoparticles are first used to generate high photo-currents electrochemically as opposed to CdS or Co3 O4 alone. Dispersed clusters of CdS nanorods conjugated with Co3 O4 nanoparticles are next shown to efficiently oxidize water and reduce methylene blue in solution.

Photosynthetic reaction center-functionalized electrodes for photo-bioelectrochemical cells.

During the last few years, intensive research efforts have been directed toward the application of several highly efficient light-harvesting photosynthetic proteins, including reaction centers (RCs), photosystem I (PSI), and photosystem II (PSII), as key components in the light-triggered generation of fuels or electrical power. This review highlights recent advances for the nano-engineering of photo-bioelectrochemical cells through the assembly of the photosynthetic proteins on electrode surfaces. Various strategies to immobilize the photosynthetic complexes on conductive surfaces and different methodologies to electrically wire them with the electrode supports are presented. The different photoelectrochemical systems exhibit a wide range of photocurrent intensities and power outputs that sharply depend on the nano-engineering strategy and the electroactive components. Such cells are promising candidates for a future production of biologically-driven solar power.