Patent Grant
10056659
Plant photosynthesis provides an unmatched quantum efficiency of nearly 100%. In recent years, significant interest has evolved in mimicking and/or harnessing the photosynthetic process for energy conversion and hydrogen generation applications. Multiple approaches to artificial photosynthesis exist, including light energy harvesting using natural pigments from plants and microorganisms and using whole cell microorganisms. Scientists have explored photosynthetic organelles such as thylakoids, chlorophyll molecules, photosystems, and oxygen evolving complexes for photo-electrochemical activity. However, the challenge of establishing electrical communication between photosynthetic reaction centers and the electrode has proven extremely difficult. Thus, to date, a photosynthetic electrochemical cell that allows direct electron transfer between the photosynthetic centers and an electrode has remained elusive.
Briefly described, embodiments of the present disclosure provide for photosynthetic electrochemical cells and methods of using the photosynthetic electrochemical cells to produce an electrical current.
In embodiments, photosynthetic electrochemical cells of the present disclosure include an anode composite having an anode, a photosynthetic reaction center (PSRC) including at least one photosynthetic compound, and a nanostructured material in electrochemical communication with the PSRC, and a cathode composite having a cathode and at least one enzyme or metallic catalyst capable of reducing O2. The PSRC is capable of oxidizing water molecules and generating electrons using a light induced photo-electrochemical reaction, and the electrons generated by the PSRC are transferred to the anode via direct electron transfer. In embodiments, the photosynthetic compounds can include, but are not limited to, PSII, PSI, plastoquinone, cyt b6f, plastocyanin, phycocyanin, phycoerythrin, carotenoids, and combinations of these compounds. In embodiments, the nanostructured material is a matrix of nanostructured materials which can be made from materials such as, but not limited to, carbon nanotubes, multi-walled carbon nanotubes, fullerenes, carbon nanoparticles, graphenes, carbon nanosheets, two-dimensional carbon platelets, other carbon nanostructured materials, metallic nanoparticles, semiconductor nanoparticles, quantum dots, and combinations of these materials.
The present disclosure also includes embodiments of photosynthetic electrochemical cells including an anode composite having an anode in electrochemical communication with a thylakoid membrane, and a cathode composite having a cathode and at least one enzyme or metallic catalyst capable of reducing O2. The thylakoid membrane in such embodiments is capable of oxidizing water molecules and generating electrons using light induced photo-electrochemical reactions, and the anode composite is configured such that electrons generated by the thylakoid membrane are conducted to the anode via direct electron transfer. In some embodiments, the thylakoid membrane is coupled to the anode by a matrix of nanostructured material, such as described above.
Additional embodiments of photosynthetic electrochemical cells of the present disclosure include an anode composite having an anode in electrochemical communication with a photosynthetic organism or a part of a photosynthetic organism, and a cathode composite having a cathode and at least one enzyme or metallic catalyst capable of reducing O2. In such embodiments, the photosynthetic organism or part thereof is capable of oxidizing water molecules and generating electrons using light induced photo-electrochemical reactions, and the anode composite is configured such that the electrons generated by the photosynthetic organism or part thereof are conducted to the anode via direct electron transfer. In some embodiments, the photosynthetic organism or part thereof is coupled to the anode by a matrix of nanostructured material, as described above. In embodiments, the photosynthetic organism can include, but is not limited to, cyanobacteria, green sulfur bacteria, algae, spirulina, chlorella, and combinations of such organisms.
The present disclosure also includes methods of generating an electrical current with a photosynthetic electrochemical cell. In embodiments, methods of generating an electrical current include providing an electrochemical cell including an anode composite having photosynthetic reaction centers (PSRC) that include at least one photosynthetic compound and are in electrical communication with an anode via a nanostructured material and a cathode composite capable of reducing O2; and exposing the electrochemical cell to light in the presence of water. In such methods, the PSRCs of the anode composite use light energy to oxidize water molecules and generate electrons, which are transferred to the anode via the nanostructured material, and then reduce O2 at a cathode, thereby inducing a potential difference between the anode and the cathode and generating an electrical current.
Embodiments of methods of generating an electrical current of the present disclosure also include converting light energy to electrical energy using a photosynthetic electrochemical cell of the present disclosure.
Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
The disclosure can be better understood with reference to the following drawings, which are discussed in the description and examples below. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.
The details of some embodiments of the present disclosure are set forth in the description below. Other features, objects, and advantages of the present disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and claims. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, organic chemistry, biochemistry, genetics, botany, electrochemistry and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps. Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. “Consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.
As used herein, the term “photosynthetic compound” includes any compound involved in the photosynthetic process, e.g., the process of harnessing light energy to induce a photochemical reaction to oxidize water molecules and generate electrons. “Photosynthetic compounds” includes “photosynthetic proteins” and protein complexes, such as, but not limited to, PSI, PSII, cyt b6f, plastocyanin, phycocyanin, and phycoerythrin as well as other non-protein, photosynthetic molecules, such as, but not limited to, plastoquinone and carotenoids. The photosynthetic compounds of the present disclosure may be isolated from the host organism and organelles in which they originate, or they may be located in a thylakoid membrane or thylakoid organelle or photosynthetic bacterial organism.
As used herein, the term “photosynthetic reaction center” (PSRC) refers to one or more photosynthetic compounds as defined above. A PSRC may include a single photosynthetic compound (e.g., PSII) or it may contain a group of photosynthetic compounds, whether isolated or working in a cluster or entity (e.g., thylakoid membrane or photosynthetic organism). A PSRC, as used in the present disclosure, has the ability to harness light energy to induce a photochemical reaction to oxidize water molecules and generate electrons.
As used in the present disclosure, two materials are in “electrochemical communication” when electrons generated by a chemical reaction of one material (e.g., photosynthetic reaction centers) can be transferred to and/or accepted by the other material (e.g., nanostructured material and/or electrode).
“Direct electron transfer,” as used in the present application indicates that an electron can be transferred to an electrode (e.g., anode) from the photosynthetic reaction center (PSRC) that catalyzed the reaction that produced the electron, as opposed to having to be transferred to the electrode by a separate shuttle molecule (e.g., a redox mediator or redox shuttle). In the present application, direct electron transfer includes the transfer of electrons generated from a photosynthetic protein to the electrode through a nanostructured material or matrix of nanostructured materials, such as where the nanostructured material couples the photosynthetic proteins, thylakoid membrane and/or photosynthetic organism to the electrode. The presence of direct electron transfer in an electrochemical cell of the present disclosure does not preclude the existence of some electron transfer occurring through a mediator, it just indicates that direct electron transfer is occurring in the cell.
As used herein, the term “anode composite” refers to a construct that provides the anode function in a photosynthetic electrochemical cell of the present disclosure. Thus, the anode composite includes the anode as well as any other materials or components coupled to the anode that provide for the oxidizing capability of the anode (e.g., nanostructured matrix material, photosynthetic reaction centers, and the like, as well as compounds or liking agents used to couple the anode to the other components of the anode composite). Similarly, the term “cathode composite” refers to a construct that includes the cathode as well as other materials that provide for the reducing activity of the cathode (e.g., the cathode and a compound capable of reducing O2, as well as any compounds or agents used to couple the cathode to the other components of the cathode composite, such as nanostructured materials and/or any linking agents).
The term “matrix of nanostructured materials”, as used in the present disclosure, includes a network or multi-dimensional structure of nanoparticles capable of coupling photosynthetic proteins, a thylakoid membrane or organelle to an electrode.
“Redox mediator” or “redox shuttle” refers to a compound capable of assisting in the transfer of electrons between a redox enzyme (e.g., a photosynthetic compound of the present disclosure that oxidizes water and generates electrons) and an electrode.
Having defined some of the terms herein, the various embodiments of the disclosure will be described.
Embodiments of the present disclosure include photosynthetic electrochemical cells capable of generating an electric current using light induced photo-electrochemical reactions catalyzed by photosynthetic compounds. The present disclosure also includes methods of generating an electrical current using photosynthetic compounds, thylakoid membranes, and/or photosynthetic bacteria or portions of photosynthetic bacteria to harness light energy and using direct electrochemical communication to transfer electrons generated by the photosynthetic proteins to an electrode.
In embodiments of the photosynthetic electrochemical cells of the present disclosure, the cell includes an anode composite that includes an anode (substrate electrode) and a photosynthetic reaction center (catalyst) including one or more photosynthetic compounds. The cell also includes a cathode or cathode composite including a cathode (substrate electrode) and at least one enzyme or metallic catalyst capable of reducing oxygen or other reductant. In embodiments of the present disclosure, the anode composite harnesses light energy to oxidize water molecules and generate electrons for transfer to the cathode for reduction of oxygen. Thus, the cathode uses electrons from the anode to reduce O2, which induces a potential difference between the anode and the cathode and generates an electrical current. The methods and the photosynthetic electrochemical cells of the present disclosure, therefore, provide a method of harnessing light energy to generate an electrical current through photo-induced electrochemical reactions and direct electron transfer.
In embodiments, the anode composite includes a photosynthetic reaction center (PSRC), or multiple PSRCs. The PSRC includes one or more photosynthetic compounds, and such photosynthetic compounds can include proteins, pigments, protein/pigment complexes, and other non-protein compounds involved in photosynthesis. In embodiments of photosynthetic electrochemical cells of the present disclosure, the PSRC of the anode composite includes at least one photosynthetic protein capable of oxidizing water molecules and generating electrons using light induced photo-electrochemical reaction. The electrons produced by the PSRCs are conducted to the anode via direct electron transfer. While not every electron produced by the PSRC will necessarily be conducted to the anode via direct electron transfer (some may be lost, and in some embodiments, a portion of the electrons generated may be transferred to the anode via a redox mediator), it will be understood that at least a portion of the electrons are conducted to the anode via direct electron transfer. In embodiments of the photosynthetic electrochemical cells of the present disclosure, the PSRC includes one or more of the following photosynthetic compounds: PSII, PSI, plastoquinone, cyt b6f, plastocyanin, phycocyanin, phycoerythrin, and carotenoids. The PSRC may include a combination of the above photosynthetic compounds. In embodiments, the PSRC of the electrochemical cell of the present disclosure includes at least one photosynthetic protein or protein complex, such as, but not limited to, PSI, PSII, cyt b6f, plastocyanin, phycocyanin, and phycoerythrin. In embodiments, the PSRC can also include non-protein photosynthetic compounds such as, but not limited to, plastoquinone and carotenoids. In embodiments, the PSRC may include one or more, two or more, three or more, or any combination of the above photosynthetic compounds. In embodiments, the anode composite may include one or more PSRCs where each PSRC may include one or more photosynthetic compounds.
The photosynthetic proteins and compounds included in the photosynthetic electrochemical cells of the present disclosure may be isolated (e.g., removed from their natural environment, organism, organelle, membrane, etc.) or they may be included in a thylakoid membrane, an in-tact thylakoid organelle, a photosynthetic organism (e.g. a photosynthetic bacterium) or a photosynthetic portion of a photosynthetic organism (e.g., a portion of the organism that is capable of photosynthesis when isolated from the source organism).
In embodiments of the photosynthetic electrochemical cells of the present disclosure, the PSRCs are included in a photosynthetic entity coupled to the anode. In some embodiments where the photosynthetic compounds are isolated from their natural environment, they can be included in a synthetic photosynthetic structure (e.g., a structure made of nanostructured materials, such as the matrix of nanostructured materials described in greater detail below). In other embodiments the photosynthetic compounds are present in a natural photosynthetic entity (e.g., a thylakoid organelle, a thylakoid membrane, a photosynthetic organism, or a portion of a photosynthetic organism). Using the proteins in a natural environment, such as a thylakoid membrane or photosynthetic bacterium, may provide certain advantages, such as facilitating the coordinated transfer of electrons between the various photosynthetic compounds in the membrane/organism. This offers various pathways for electron transfer between the photosynthetic compounds in the PSRCs and the anode, rather than just a single path offered by a single isolated photosynthetic protein. Thus, in some embodiments it may be advantageous to utilize natural photosynthetic entities, such as a portion of a thylakoid membrane, an intact thylakoid organelle, or photosynthetic organism, or part thereof in the electrochemical cells of the present disclosure.
As used herein, a PSRC can refer to an isolated photosynthetic compound, to a grouping or cluster of photosynthetic compounds working together, to a synthetic structure including a cluster of photosynthetic compounds, to the photosynthetic compounds or groups of compounds within such a synthetic structure, to a single photosynthetic entity such as a thylakoid membrane, thylakoid organelle or photosynthetic organism, or to a group of photosynthetic compounds within such a photosynthetic entity. Thus, a PSRC refers to any single or grouping of photosynthetic compounds capable of oxidizing water molecules and generating electrons using light induced photo-electrochemical reactions.
The photosynthetic compounds (whether isolated or part of a natural or synthetic photosynthetic structure) are included in the anode composite such that the PSRCs are in electrochemical communication with the anode so that electrons generated during photosynthetic reactions can be conducted directly to the anode. In some embodiments, the PSRCs are coupled to the anode by a nanostructured material. In such embodiments, the anode composite also includes a nanostructured material in electrochemical communication with at least one PSRC and the anode. In embodiments where the PSRCS are isolated photosynthetic compounds or clusters of isolated compounds, the PSRCs may be coupled to and/or integrated into a nanostructured material such that they form a synthetic photosynthetic structure, as described above, and the photosynthetic structure can be coupled directly to the anode. In other embodiments, the PSRCs may be a natural photosynthetic entity, and the PSRC/entity may be coupled to the anode via a matrix of nanostructured material. In some embodiments, the anode can be functionalized with a nanostructured material, and the PSRC is in electrochemical communication with the nanostructured material of the anode.
In embodiments, the nanostructured material is a matrix of nanostructured material made of a material capable of being coupled to and in electrochemical communication with the PSRCs. In embodiments, the nanostructured materials include, but are not limited to, carbon based nanomaterials, metallic nanoparticles, semiconductor nanoparticles, quantum dots or combinations of these materials. Some embodiments of carbon based nanomaterials useful for the electrochemical cells of the present disclosure include, but are not limited to, materials such as carbon nanotubes, multi-walled carbon nanotubes, fullerenes, carbon nanoparticles, graphenes, two dimensional carbon nanosheets, graphite platelets, and the like. In some specific embodiments, the matrix of nanostructured material is multi-walled carbon nanotubes.
In the electrochemical cells of the present disclosure, in embodiments, the PSRCs are coupled to the matrix of nanostructured material in the anode by a cross-linking agent, such as, but not limited to, 1-pyrenebutanoic acid succinimidyl ester (PBSE) or other protein homo- or hetero-bifunctional cross-linking agent.
Although the methods and photosynthetic electrochemical cells of the present disclosure allow for direct electron transfer between the PSRCs and the anode, in some instances it may be advantageous to include a redox mediator (also known as a redox shuttle) to facilitate transfer of electrons between the photosynthetic proteins and the nanostructured material/anode. In embodiments the redox mediator may be chosen from mediators such as, but not limited to, ferricyanide, quinone-based compounds, osmium complex based compounds, any other redox chemical compound and combinations of the above.
In embodiments of the photosynthetic electrochemical cells of the present disclosure, the cathode includes at least one compound capable of reducing a reductant, such as, but not limited to O2, ferro/ferricyanide couple, and the like. In embodiments, the photosynthetic electrochemical cells of the present disclosure include a cathode composite including a cathode and a compound or combination of compounds capable of reducing a reductant, such as O2. In embodiments, the cathode composite may also include a nanostructured material to facilitate electrochemical communication between the cathode and the oxygen-reducing compounds. The nanostructured material of the cathode composite can be any of the nanostructured materials described above in reference to the anode composite. In embodiments, the compound capable of reducing O2 can include, but is not limited to, an enzyme or a metallic catalyst. In some embodiments the enzyme capable of reducing O2 can include, but is not limited to, laccase, bilirubin oxidase, ascorbate oxidase, tyrosinase, catechol oxidase, and combinations of these or other enzymes. In embodiments the enzyme capable of reducing O2 include a metallic catalysts such as, but not limited to platinum, silver, gold, cobalt, nickel, iron and combinations of these metals. In embodiments, the compound capable of reducing ferro/ferricyanide couple, can include but is not limited to, could be a metal, semiconductor or carbon or a chemical capable of reducing the ferro/ferricyanide couple.
In embodiments of the present disclosure the anode and cathode can be made of any standard electrode material, such as carbon, metals, semiconductor such as silicon, and the like. One advantage to the cells of the present disclosure is that the use of the nanostructured materials to couple the photosynthetic compounds or oxygen reducing compounds to the electrodes allows coupling of the photosynthetic compounds to the electrode without the need for expensive precious metal electrodes (e.g., gold, silver, platinum, etc.) and without complex immobilization procedures that are incompatible with other, less expensive electrode materials such as carbon.
The examples below and the attached figures provide additional detail regarding some embodiments of the photosynthetic electrochemical cells of the present disclosure and the elements of the anode and cathode described above.
The methods of the present disclosure include methods of generating an electrical current including using the photosynthetic electrochemical cells of the present disclosure to convert light energy to electrical energy. In embodiments, the methods of the present disclosure include using thylakoid membrane photosynthetic proteins and compounds and/or photosynthetic bacterial proteins and compounds to harness light energy to oxidize a water molecule and directly transfer electrons from the photosynthetic proteins to an anode using direct electrochemical communication. In embodiments, methods of the present disclosure also include using the electrons from the anode to reduce O2 at a cathode, thereby inducing a potential difference between the anode and the cathode and generating an electrical current.
Briefly described, some methods of generating an electrical current according to the present disclosure include providing an electrochemical cell that has an anode composite that includes a PSRC in electrical communication with an anode via a nanostructured material and a cathode composite capable of reducing oxygen. The electrochemical cell is exposed to light in the presence of water so that the PSRC uses light energy to oxidize water molecules and generate electrons, which are transferred to the anode via the nanostructured material, flow to the cathode, where they reduce oxygen, thereby inducing a potential difference between the anode and the cathode to generate an electrical current.
In embodiments the present disclosure includes methods of generating an electrical current by providing a photosynthetic electrochemical cell of the present disclosure and exposing the photosynthetic electrochemical cell to light in the presence of water, such that the PSRCs use light energy to oxidize a water molecule and generate electrons which are transferred to the anode via the nanostructured material, and the electrons generated at the anode flow to the cathode where they are used to reduce O2 at a cathode, thereby inducing a potential difference between the anode and the cathode and generating an electrical current. The photosynthetic electrochemical cells used in the methods of the present disclosure can include an anode composite having photosynthetic reactions centers in electrical communication with an anode via a nanostructured material as described above and include a cathode or cathode composite capable of reducing O2 or other reductant as described above.
Now having described the embodiments of the present disclosure, in general, the Examples, below, describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Spinach thylakoids were coupled to electrodes via multiwalled carbon nanotubes using a molecular tethering chemistry. The resulting thylakoid-carbon nanotube composite showed high photo electrochemical activity under illumination. It is believed to be the first time multiple membrane proteins have been observed to participate in direct electron transfer with the electrode, resulting in the generation of photocurrents. Thus, it is believed that the present disclosure describes the first of its kind for natural photosynthetic systems.
The high electrochemical activity of the thylakoid-MWNT composites has significant implications for both photosynthetic energy conversion and photofuel production applications. A fuel cell type photosynthetic electrochemical cell developed using thylakoid-MWNT composite anode and laccase cathode produced a maximum power density of 5.3 μW cm−2 comparable to that of enzymatic fuel cells. The carbon based nanostructured electrode has the potential to serve as an excellent immobilization support for photosynthetic electrochemistry based on the molecular tethering approach as demonstrated in the present example.
Introduction
Plant photosynthesis has evolved over 2.5 billion years to convert solar energy into chemical energy using only water, with an unmatched quantum efficiency of nearly 100%(1, 2). In recent years, there has been an increasing interest in mimicking the natural photosynthetic process for energy conversion and photo fuel (ethanol, H2 etc.) production(3-5). This is being carried out using synthetic routes such as metal oxides, semiconductors or chemical catalysts for carrying out the light-driven water splitting reaction(6-11). Alternatively, components of the naturally occurring photosynthetic apparatus of bacteria(12), algae and plants(13-16) have been employed for bioconversion applications. For example, the direct conversion of light into electricity based on photosynthesis in an electrochemical cell has been investigated in the past(17-20), using natural systems such as thylakoids, chlorophyll molecules, photosynthetic reaction centers(21-28), and even whole cells such as cyanobacteria(29-31). Besides these representative attempts, the low electron transfer efficiency of the photosynthetic machinery to the electrodes still plague the power output performance of these systems. Isolated photosynthetic components systems possess some advantages over whole cells, such not needing nutrients for sustenance, and not having competition between respiration and photosynthesis in sharing the electron transfer pathways. However when isolated plant photosynthetic systems have been used on electrodes they have suffered from low efficiencies due biomolecule stability, improper immobilization, lack of electrical communication, etc(10). For light energy harvesting applications, it is thermodynamically advantageous to collect electrons directly from the molecules at high-energy states along the photosynthetic electron transport pathway, such as an excited photosystem II (PSII)(26,32,33). Moreover, for direct light-electricity conversion applications, it is generally preferable to use a higher order plant based system that uses only water as the electron donor such as PSII, rather than isolated PSI complexes, which require an alternate electron donor. For this purpose, attempts have been made to immobilize PSII reaction centers on to the electrode using cytochromes(34) or nickel-nitrilotriacetic acid(35) as cross-linkers or through some terminal electron acceptors such as CoIII complexes(36). All these methods use precious metals (e.g., gold) as the immobilization support and use expensive immobilization procedures that cannot be extended to other electrode materials (e.g., carbon). Accordingly, the precious metal based electrochemistry carries less practical value for energy conversion applications.
On the other hand, photosynthetic organelles or membranes also possess advantages over isolated reaction center complexes for electrochemical applications. Some such advantages include: high individual protein stability(37), the ability to use simpler immobilization procedures, and the presence of multiple electron transfer routes. For example, if thylakoid membranes are used in the place of isolated PSII complex, then the electron transfer from an oxygen evolving complex (OEC) site to the electrode can be achieved via plastoquinone, cytochrome (cyt) b6f, plastocyanin, ferredoxin, PSI, etc. in addition to a direct transfer from PSII(34). Moreover retention of their natural partners results in enhanced stability of the individual proteins in thylakoids in comparison to their isolated counterparts(38). Therefore using thylakoids as photo-biocatalysts or otherwise complexing photosynthetic proteins within a photosynthetic structure offers the potential for high photo-electrochemical activity as well as high stability for both energy conversion and fuel production applications.
The present example demonstrates the photo-electrochemical activity of spinach thylakoids immobilized on to multi-walled carbon nanotube (MWNT) modified electrodes. By employing a carbon-based material, the necessity for expensive precious metal-based catalyst supports to immobilize photosynthetic machinery onto electrode surfaces was eliminated. The molecular tethering approach described in the present example by using nanostructured materials to couple the photosynthetic structure to the electrode helps establish multiple attachments between the thylakoid membranes and the electrode surface. Moreover, the present example demonstrates that by using the entire membrane instead of isolated photosystem complexes, manipulation of the electron transfer pathways to achieve high electron transfer flux for photo current generation was possible. This example also demonstrates direct light to energy conversion with water as the only input using a photosynthetic fuel cell composed of a thylakoid based anode and laccase based cathode, first of its kind employing a plant photosynthetic membrane and an enzymatic cathode operating at neutral pH. A schematic of the thylakoid membranes immobilized on MWNT modified electrode and the associated electron transport pathways are shown conceptually in
Materials
Thylakoids were extracted from fresh organic spinach obtained from local market. MWNT, 10 nm diameter and 1-2 μm length (Dropsens, Spain) was used as the immobilization support for the thylakoids. 1-pyrenebutanoic acid succinimidyl ester (PBSE) (Anaspec) was used as the molecular tethering agent to attach thylakoids on MWNT. Potassium ferricyanide, redox mediator, and N,N-dimethyl formamide (DMF), solvent used for reagent preparation, were purchased from Acros Organics. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was purchased from Tokyo Chemical Industry. Laccase from Trametes versicolor (Sigma) was the enzyme used on the cathode. Potassium cyanide (KCN) was purchased from Fisher Scientific. Paraquat/Diquat was purchased from Ultra Scientific. Tricine (OmniPur), sorbitol (EMD Chemicals Inc), ethylenediaminetetraacetic acid (EDTA) (VWR), and potassium hydroxide (Mallinckrodt Baker) were used for preparing buffer solutions. Phosphate buffer for electrochemical testing was prepared using monobasic and dibasic potassium phosphates (VWR). All buffers were prepared using nanopure distilled water (ddH2O). Electrolyte (buffer) solutions were purged for 30 min with N2 to remove any dissolved O2.
Methods
Thylakoids were isolated from Spinacia oleracea (spinach) leaves based on the procedure given in literature(39) using a refrigerated centrifuge (Beckman Coulter Avanti J-E). The procedure is known to produce a mixture of both intact organelles and broken thylakoid membranes. During isolation the chlorophyll concentration was determined to be between 2.5 and 3.0 mg mL−1 (average of 2.8 mg mL−1) via UV-Vis absorbance measurements using a spectrophotometer (UV-Vis) (Cary Varian 50 Bio, Sparta, N.J.) using the formula given in the
Slurries of MWNT were prepared by dispersing 1 mg mL−1 of MWNTs in 10 mM DMF by 10 min ultra sonication using an ultrasonic homogenizer (Omni International) at the power output of 20 watts. The dispersion was sonicated again for 1 h in a bath sonic cleaner (XP-Pro). The obtained MWNT dispersion was used as it is for electrode modifications.
A molecular tethering approach was used to immobilize thylakoid membranes on the carbon nanotube matrix using PBSE as the linker (e.g., tethering agent), which has been demonstrated to produce excellent bio-electrochemical characteristics(40-42). In this method, first the electrodes (0.02 cm2) were polished with 0.05 μm alumina slurry. The polished electrode was rinsed and ultrasonicated in ddH2O for 8 min. Then the electrode was modified with 4 μL of MWNT dispersion and later dried at 70° C. After drying, a desired volume of 10 μM PBSE was drop casted on the MWNT modified electrode and incubated for 15 min in an ice bath. The resulting modified electrode was washed first with DMF to remove the loosely bounded PBSE and then with tricine buffer to neutralize the pH of the electrode surface. Finally, 5 μL of thylakoid suspended solution (corresponds to 0.44 μg cm2 chlorophyll loading) was drop casted on the electrode surface and incubated for 1 h in the dark in an ice bath. The modified electrode was then washed with tricine buffer prior to experimentation.
Testing
Bare or thylakoid modified MWNT was used as the working electrode in a 3-electrode electrochemical cell setup with a platinum wire counter electrode (Alfa Aesar) and a silver-silver chloride (Ag/AgCl) reference electrode (CH Instruments). All electrochemical experiments were conducted at 25±2° C. using 0.1 M tricine buffer pH 7.8 as the electrolyte. Electrochemical tests were performed both in the presence and absence of [Fe(CN)6]3−/4− as a redox mediator. The operating conditions for electrochemical tests were chosen based on a series of optimization tests for thylakoid loading, immobilization duration, mediator concentration and anode potential over a reasonable range, the results of which are given in
Chlorophyll Concentration Measurements
The chlorophyll concentration (Cch) was calculated by using the data from UV-Vis spectrum into the equation (E1).
Optimization of Composite Composition
The plots of current versus time at fixed anode potentials were used as a guiding tool for optimizing the thylakoid-MWNT composite electrode (
Results and Discussion
Physical Characterization
Tapping mode AFM and SEM were used to study the morphology of thylakoid-modified MWNT electrodes. The AFM topography, amplitude and phase images of the unmodified MWNT (control) and thylakoid-modified MWNT electrodes are shown in
Electron Transfer Pathways in Thylakoids
The thylakoid membrane consists of several integral membrane proteins that could partake in in electron transfer to the electrode. As schematically depicted in
In addition to the three ETPs discussed above, other routes for electrochemical communication between thylakoids and MWNT electrode are possible. For example, a direct electron transfer from plastoquinone site of PSII to MWNT is possible if the stromal side of PSII complex is orientated towards the electrode. However the present experimental results (discussed in the following paragraphs) indicated no significant contribution from any additional routes to the electrochemical charge transfer and hence were not depicted in
Redox Electrochemistry of Immobilized Thylakoid Membranes
Direct Electron Transport
Cyclic voltammetry was used to study redox activity of the unmodified and thylakoid-MWNT composite modified electrodes and to verify the existence of the ETPs discussed above in
Mediated Electron Transport
In a separate set of experiments, 1.5 mM [Fe(CN)6]3−/4− couple was added to the electrolyte to assist electron transfer from thylakoid membrane to the MWNT electrode. Ferricyanide is a suitable choice because of its minimal photo-activity(50,51), compared to benzoquinone mediators used elsewhere(52), as confirmed in separate studies (see
Photo-Electrochemical Activity of Thylakoid-MWNT Composites
The photo-electrochemical activity of thylakoid modified MWNT electrodes were evaluated using open circuit potential (OCP), potentiostatic polarization, and AC impedance measurements.
Open Circuit Potentials
Potentiostatic Polarization
The photo-electrochemical activity of the thylakoid-MWNT composite was evaluated at constant anode potential of 0.2 V and the variation of anode current with time was evaluated during light on-light off cycles. The photocurrent densities ranged from 30 to 70 μA cm−2 during initial cycles eventually attaining steady state within ≈400 s in the range of 23 to 38 μA cm−2 that lasted for 1 week. (
aSAM—self assembled monolayer; RC—reaction center; RBS—Rhodobacter sphaeroides
bPhotocurrent density was calculated using the photocurrent and the active surface area provided in the respective literature
The thylakoid-MWNT composite was also found to be very responsive to the light intensity (see
The decrease in the amplitude of photo-currents over continuous light on-off cycles observed in
AC Impedance
AC impedance studies were also carried out on thylakoid-MWNT composites under light and dark conditions in the presence of mediator to understand the influence of individual resistances on photocurrent generation. The Nyquist plots (−Z″ vs. Z′) of the impedance data and the equivalent electrical circuit (to which the data was fitted for parametric analysis) are given in
An impedance observation in the absence of mediator also showed high charge transfer impedance indicating this system was not limited by the mediator diffusion. Separate measurements performed on thylakoid modified electrodes with and without MWNT platform indicated lower ohmic resistance (after eliminating the solution impedance contribution) for the MWNT electrodes. This confirmed the existence of high electronic conductivity for the thylakoid-MWNT composite electrodes.
Role of PSII vs. PSI in Photocurrent Generation
In order to confirm that the light-induced water splitting reaction is the electron source for the observed photocurrents in our composite electrodes, two control experiments were performed. The first control experiment was aimed at studying the effect of blocking the PSII reaction center complex and the second was aimed at blocking the PSI reaction center complex from participating in the photosynthetic electron transport. For inhibiting PSII activity, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added to the thylakoid solution prior to immobilization. DCMU is a herbicide that specifically blocks the QA→QB site in PSII complex, severing the electron transport from PSII to the subsequent proteins in the pathway(59). As shown in
Thylakoid-Laccase Photosynthetic Electrochemical Cell
A fuel cell type electrochemical cell was constructed using the thylakoid-MWNT composite anode and laccase-MWNT composite cathode and tested in an electrolyte solution (PBS buffer pH 6.8). The anode oxidizes water upon illumination with light using thylakoid-MWNT composites as photo-biocatalysts, whereas the cathode reduces oxygen to regenerate water in the system using laccase as an enzymatic bio-electrocatalyst. The use of laccase for bio-electrocatalytic oxygen reduction in biological fuel cells has been well established(40-42). The molecular tethering approach used for thylakoid immobilization was also used for laccase immobilization on MWNT at the cathode. The open circuit potential of cell was ˜0.4 V. Polarization tests were performed under illumination, at constant applied potentials between 0.35 V and 0 V and the resulting steady state currents were measured and shown in
Inhibition of Plastocyanin by KCN:
The thylakoid-MWNT composites prepared using KCN in the immobilization mixture exhibited a significant reduction the plastocyanin activity by up to 73%. Although KCN only inhibits plastocyanin, the redox activity of cyt b6f (peak at 0 V) was also reduced by 23%. This could be due to the lack of an electron acceptor for cyt b6f (when plastocyanin was inhibited), which may result in an excited cyt b6f (electron rich) that reacts with oxygen to form peroxides that degrade the cyt b6f activity over time as suggested by Fuerst et. al. (E. P. Fuerst and M. A. Norman, Interaction of herbicides with photosynthetic electron transport. Weed Science 39, 458-464 (1991)).
Photoactivity of Mediators:
Constant potential measurements on unmodified MWNT electrodes in the presence of mediators at 0.2 V under light on-off conditions showed that the ferricyanide [Fe(CN)6]3−/4− redox couple exhibited less photo-response than the benzoquinone complexes used by others in the literature. Therefore the observed photocurrent activity in our thylakoid-MWNT composite electrodes can be directly attributed to thylakoids and not the [Fe(CN)6]3−/4− mediator.
Absorption Spectra of Mediators and Inhibitors:
The peak at 673 nm indicates absorbance via chlorophyll-a. The absorption spectrum for the thylakoid-free MWNT electrode did not contain the chlorophyll peak (
Effect of Light Intensity on Photocurrent:
The photo-electrochemical response of the thylakoid-MWNT composite modified electrodes varied with light intensity as shown in
Advantages of Thylakoid Immobilization:
To understand if there are advantages associated with immobilizing thylakoids for carrying out photo-electrochemical reactions, rather than suspending them in the solution, separate experiments were performed.
Steady State Analysis:
When light was illuminated a large increase in anodic current was observed due to ferrocyanide oxidation at the electrode surface. This would require a continuous ferricyanide reduction by thylakoid membrane proteins in the presence of light. Over time the current generation stabilized to a constant value at approximately 0.675 μA (
Herbicide Inhibition of Photosystem II:
Upon exposing the thylakoid to DCMU herbicide that inhibits PSII activity, no photo-electrochemical activity was noticed in the light on-off tests. However, the cyclic voltammograms under light showed no loss in the redox activities of both cyt-b6f and plastocyanin (
Non-Involvement of PSI in Photocurrent Generation
The non-involvement of PSI in photocurrent generation was studied by inhibiting PSI activity by paraquat/diquat solution mixture (of the bipyridillum family). The solution mixture was added to thylakoids prior to immobilization. Paraquat (E0=−0.45 V) acts as a competitor to ferredoxin (FD, E0=−0.51 V) for accepting the electrons from the Fa/Fb site of PSI (E0=−0.56 V) in the photosynthetic pathway. As shown in
The present example demonstrated high photo-electrochemical activity of immobilized thylakoid-MWNT composites for light energy harvesting application. The findings have significant implications for photosynthetic energy conversion and photo fuel production. The composites exhibited direct electron transfer activity, which can be enhanced by using a suitable mediator. Control experiments confirmed that the light-induced water splitting reaction at the PSII complex was the primary source of electrons for photo-current generation. At least some advantages of using thylakoid membranes as opposed isolated photosystems lie in the self-assembly and utilization of direct electrochemical redox activities of more than one membrane proteins present in the thylakoid. The thylakoid-MWNT composite electrode yielded a maximum current density of 68 μA cm−2 and a steady state current density of 38 μA cm−2, which are two orders of magnitude higher than the previously reported values in other systems (Table 1). The photo-electrochemical cell delivered a maximum power output of 5.3 μW cm−2. No optimization efforts to enhance the power density were attempted in this work. Accordingly, improvements in power densities can be realized by engineering optimization such as, but not limited to, designing suitable membrane-less electrochemical cells, selecting materials for electrode substrates, developing superior immobilization methods etc. Additional understanding of the electron transport pathways will help enhance direct electron transfer and development of a mediator-free system to demonstrate direct light to electricity conversion. The bio-inspired photosynthetic energy conversion technology using plant thylakoids demonstrated in this example offers great potential for green energy harvesting based on a natural process that evolved over millions of years.
References, each of which is incorporated herein by reference.
In the present example, electrochemical cells were designed and tested similar to those described in Example 1, above, except in place of thylakoids, photosynthetic bacterial organism were complexed with carbon nanotubes to provide photosynthetic energy conversion.
Materials and Methods:
Materials and methods are similar to those described in Example 1 above, except as noted below. Sterile cultures of Nostoc sp. and AV were obtained from Bioconversion Research Centre, UGA and cultured in our laboratory in shake flasks using BG11 medium under 12 hr light/dark cycles. Once the optical density at 750 nm (OD750) reaches around 1 (happens ˜15 after the culture inoculation), the culture was harvested by centrifuging at 5000 rpm for 10 min at room temperature and washed in phosphate buffer (0.1 M, pH 7) before used for immobilization onto the electrode. 5 μl of multi-walled carbon nanotubes suspension (1 mg/ml in DMF) was dropped on the carbon paper and allowed to dry. 5 μl of the washed bacterial cells were immobilized on the top of carbon nanotube layer, allowed to air dry. The resulting bacterial cell modified carbon nanotube electrode was then used for the photo-electrochemical experiments.
Results and Discussion:
The morphology of the immobilized Nostoc sp. and Anabaena variabilis (AV) on the multi-walled carbon nanotubes (MWNT) were studied by scanning electron microscopy (SEM).
In general, the value of open circuit potential (OCP) is dictated by the mixed potential caused by a variety of electron transfer reactions whose individual redox potentials range from −1.3 to 1.0 V vs. SHE.
The photo-activity of the photosynthetic bacteria modified electrodes were evaluated at constant working electrode potential of 0.2 V and the corresponding photo-current response was measured over time during light on-off cycles.
The current vs. time plots of fixed anode potentials were used as a tool for optimizing the photosynthetic bacteria loading on the electrode surface. The results showed that the photocurrent density was directly proportional to Nostoc sp. loading (
In the case of AV (
The photo-electrochemical response of the Nostoc sp. varied with incident light intensity as shown in
The stability of current generation was studied by observing the steady state current change during the experiments. To identify the steady state, currents were measured in continuous light and dark conditions without cycling.
The major pigment present in all known photosynthetic organisms is chlorophyll a, which forms the reaction centers in both PSII (P680) and PSI (P700), absorbing light efficiently at 465 nm and 665 nm. Additionally, cyanobacteria such as AV and Nostoc also possess certain accessory pigments such as phycocyanin, phycoerythrin and carotenoids that maximize the range of action spectrum by absorbing a range of wavelengths other than that absorbed by Chlorophyll a (Chl a). Upon absorbing the characteristic light, these accessory pigments transfer the absorbed energy to other pigments and finally to the reaction center Chl a.
The mechanism of electron transfer from the photosynthetic electron transfer chain (PETC) to the MWCNT was also studied with the help of photosystem inhibitors. Inhibitors such as DCMU ((3-(3,4-dichlorophenyl)-1,1-dimethylurea), DBMIB (2,5-dibromo-3-methyl-6-isopropylbenzoquinone), KCN (potassium cyanide) and antimycin-A specifically block a particular site of the electron transfer chain (
Experiments were conducted to measure the photocurrent produced by the Nostoc sp. after incubating the cells with the inhibitors such as DCMU, DBMIB and KCN at varying concentrations, and the results have been summarized in
The inhibition by DBMIB is highly dependent on the concentration of the DBMIB used as illustrated in
Inhibition of photocurrent by KCN is considerable (
The Q cycle catalyzed by Q0 and Qi sites of Cyt b6f complex represent another possible source for the photocurrent. The two electrons coming from the oxidation of PQH2 at Q0 site of Cyt b6f complex are not completely transferred to PC; rather only one electron is transferred through Cyt f to PC, and the other electron is used to reduce PQ at Qi site through atypical heme x (Zhang et al, 2004; Stroebel et al, 2003). It has been investigated that this heme x near the Qi site functions as a redox wire allowing ferredoxin or other electron carrier to reduce PQ pool.
The foregoing examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is in atmospheres. Standard temperature and pressure are defined as 25° C. and 1 atmosphere.
It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term “about” can include traditional rounding according to measurement techniques and the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.
Many variations and modifications may be made to the embodiments described in the preceding Examples. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
This application is a Divisional of co-pending US application having Ser. No. 13/957,494, filed on Aug. 2, 2013. This application also claims priority to U.S. provisional application entitled “Photosynthetic Electrochemical Cells”, having Ser. No. 61/679,118 filed on Aug. 3, 2012, which is entirely incorporated herein by reference.
| Number | Date | Country |
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| WO2010132633 | Nov 2010 | WO |
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| 20160380321 A1 | Dec 2016 | US |
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| 61679118 | Aug 2012 | US |
| Number | Date | Country | |
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| Parent | 13957494 | Aug 2013 | US |
| Child | 15257091 | US |
