US 20140O38065A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0038065 A1 Ramasamy (43) Pub. Date: Feb. 6, 2014
(54) PHOTOSYNTHETIC ELECTROCHEMICAL Publication Classification CELLS (51) Int. Cl. (71) Applicant: University of Georgia Research HIM I4/00 (2006.01) Foundation, Inc., Athens, GA (US) (52) U.S. Cl. CPC ...... H0IM 14/005 (2013.01) (72) Inventor: Ramaraja P. Ramasamy, Watkinsville, USPC ...... 429/401; 429/532 GA (US)
(22) Filed: Aug. 2, 2013 O O The present disclosure provides photosynthetic electro Related U.S. Application Data chemical cells including photosynthetic compounds and (60) Provisional application No. 61/679,118, filed on Aug. methods of generating an electrical current using the photo 3, 2012. synthetic electrochemical cells.
Patent Application Publication Feb. 6, 2014 Sheet 1 of 28 US 2014/003.8065 A1
Patent Application Publication Feb. 6, 2014 Sheet 2 of 28 US 2014/003.8065 A1
Tophography Amplitude Phase
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FIG. 2 Patent Application Publication Feb. 6, 2014 Sheet 3 of 28 US 2014/003.8065 A1
FIG. 3A FIG. 3B
FIG. 3C Patent Application Publication Feb. 6, 2014 Sheet 4 of 28 US 2014/003.8065 A1
FIG. 4A
FIG. 4B
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-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 Potential (V vs. Ag|AgCl) Patent Application Publication Feb. 6, 2014 Sheet 5 of 28 US 2014/003.8065 A1
FIG. 5A 0.30 0.20 0.15 0.10 0.05 x-Thylakoid. MWNT xx - Unmodified MWNT
100 300 500 700 900 1100 1300 Time (s)
FIG. 5B «Thylakoid - MWNT - Unmodified MWNT
125 200 275 350 425 500 575 Time (s) Patent Application Publication Feb. 6, 2014 Sheet 6 of 28 US 2014/003.8065 A1
FIG.5C 140 120 100
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(a) x 82.68 gig (b) 13.8 big (c) & 6.89 kg (d) 4.13:g (e) : y-(b)
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Time (s) Patent Application Publication Feb. 6, 2014 Sheet 10 of 28 US 2014/003.8065 A1
FIG. 9C - O. SS (a) - 8: 100 M (b) 200 M O.
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Patent Application Publication Feb. 6, 2014 Sheet 11 of 28 US 2014/003.8065 A1
«Thy Controi xxy + KCN
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400 500 600 700 800 Wavelength (nm) Patent Application Publication Feb. 6, 2014 Sheet 13 of 28 US 2014/003.8065 A1
FIG. 12C xx: Para/Diduat : ... Thy+Para/Diquat
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100 soo 600 700 800 Wavelength (nm) Patent Application Publication Feb. 6, 2014 Sheet 14 of 28 US 2014/003.8065 A1
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FIG. 16A 8. xxxx terticit: ~ 888 orbicide
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EIV vs. Aglagol Patent Application Publication Feb. 6, 2014 Sheet 18 of 28 US 2014/003.8065 A1
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FIG. 18 Patent Application Publication Feb. 6, 2014 Sheet 20 of 28 US 2014/003.8065 A1
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FIG. 28 Patent Application Publication Feb. 6, 2014 Sheet 28 of 28 US 2014/003.8065 A1
FIG. 29A
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PHOTOSYNTHETIC ELECTROCHEMICAL cal reactions, and the anode composite is configured such that CELLS electrons generated by the thylakoid membrane are con ducted to the anode via direct electron transfer. In some CROSS-REFERENCE TO RELATED embodiments, the thylakoid membrane is coupled to the APPLICATION anode by a matrix of nanostructured material. Such as described above. 0001. This application claims priority to copending U.S. 0006 Additional embodiments of photosynthetic electro provisional application entitled, “Photosynthetic electro chemical cells of the present disclosure include an anode chemical cells.” having Ser. No. 61/679.118, filed Aug. 3, composite having an anode in electrochemical communica 2012, which is entirely incorporated herein by reference. tion with a photosynthetic organism or a part of a photosyn thetic organism, and a cathode composite having a cathode BACKGROUND and at least one enzyme or metallic catalyst capable of reduc 0002 Plant photosynthesis provides an unmatched quan ing O. In such embodiments, the photosynthetic organism or tum efficiency of nearly 100%. In recent years, significant part thereof is capable of oxidizing water molecules and interest has evolved in mimicking and/or harnessing the pho generating electrons using light induced photo-electrochemi tosynthetic process for energy conversion and hydrogen gen cal reactions, and the anode composite is configured such that eration applications. Multiple approaches to artificial photo the electrons generated by the photosynthetic organism or synthesis exist, including light energy harvesting using part thereof are conducted to the anode via direct electron natural pigments from plants and microorganisms and using transfer. In some embodiments, the photosynthetic organism whole cell microorganisms. Scientists have explored photo or part thereof is coupled to the anode by a matrix of nano synthetic organelles such as thylakoids, chlorophyll mol structured material, as described above. In embodiments, the ecules, photosystems, and oxygen evolving complexes for photosynthetic organism can include, but is not limited to, photo-electrochemical activity. However, the challenge of cyanobacteria, green Sulfur bacteria, algae, spirulina, chlo establishing electrical communication between photosyn rella, and combinations of Such organisms. thetic reaction centers and the electrode has proven extremely 0007. The present disclosure also includes methods of difficult. Thus, to date, a photosynthetic electrochemical cell generating an electrical current with a photosynthetic elec that allows direct electron transfer between the photosyn trochemical cell. In embodiments, methods of generating an thetic centers and an electrode has remained elusive. electrical current include providing an electrochemical cell including an anode composite having photosynthetic reaction SUMMARY centers (PSRC) that include at least one photosynthetic com 0003 Briefly described, embodiments of the present dis pound and are in electrical communication with an anode via closure provide for photosynthetic electrochemical cells and a nanostructured material and a cathode composite capable of methods of using the photosynthetic electrochemical cells to reducing O, and exposing the electrochemical cell to light in produce an electrical current. the presence of water. In such methods, the PSRCs of the 0004. In embodiments, photosynthetic electrochemical anode composite use light energy to oxidize water molecules cells of the present disclosure include an anode composite and generate electrons, which are transferred to the anode via having an anode, a photosynthetic reaction center (PSRC) the nanostructured material, and then reduce O at a cathode, including at least one photosynthetic compound, and a nano thereby inducing a potential difference between the anode structured material in electrochemical communication with and the cathode and generating an electrical current. the PSRC, and a cathode composite having a cathode and at 0008 Embodiments of methods of generating an electrical least one enzyme or metallic catalyst capable of reducing O. current of the present disclosure also include converting light The PSRC is capable of oxidizing water molecules and gen energy to electrical energy using a photosynthetic electro erating electrons using a light induced photo-electrochemical chemical cell of the present disclosure. reaction, and the electrons generated by the PSRC are trans 0009. Other systems, methods, features, and advantages ferred to the anode via direct electron transfer. In embodi of the present disclosure will be or become apparent to one ments, the photosynthetic compounds can include, but are not with skill in the art upon examination of the following draw limited to, PSII, PSI, plastoquinone, cyt bef, plastocyanin, ings and detailed description. It is intended that all Such phycocyanin, phycoerythrin, carotenoids, and combinations additional systems, methods, features, and advantages be of these compounds. In embodiments, the nanostructured included within this description, and be within the scope of material is a matrix of nanostructured materials which can be the present disclosure. made from materials such as, but not limited to, carbon nano BRIEF DESCRIPTION OF THE DRAWINGS tubes, multi-walled carbon nanotubes, fullerenes, carbon nanoparticles, graphenes, carbon nanosheets, two-dimen 0010. The disclosure can be better understood with refer sional carbon platelets, other carbon nanostructured materi ence to the following drawings, which are discussed in the als, metallic nanoparticles, semiconductor nanoparticles, description and examples below. The components in the quantum dots, and combinations of these materials. drawings are not necessarily to scale, emphasis instead being 0005. The present disclosure also includes embodiments placed upon clearly illustrating the principles of the present of photosynthetic electrochemical cells including an anode disclosure. composite having an anode in electrochemical communica 0011 FIG. 1 illustrates a schematic representation of a tion with a thylakoid membrane, and a cathode composite thylakoid membrane tethered to MWNT modified electrode having a cathode and at least one enzyme or metallic catalyst using PBSE linkers and the likely electron transportpathways capable of reducing O. The thylakoid membrane in Such (a), (b) and (c) between thylakoid membrane proteins and embodiments is capable of oxidizing water molecules and the electrode. OEC, PQ, Cyt, PC, FD, and ATP Syn represent generating electrons using light induced photo-electrochemi oxygen evolving complex, plastoquinone, cytochrome, plas US 2014/003 8065 A1 Feb. 6, 2014 tocyanin, ferredoxin and ATP synthase, respectively. PSI and (0023 FIGS. 12A-12D illustrate the absorption spectrum PSII represent the photosynthetic reaction centers I and II, of thylakoid membranes with exposure to ferricyanide media respectively. Other components of the thylakoid membrane tor (FIG. 12A), KCN (FIG. 12B), paraquat/diquat (FIG. are not shown. 12C), and DCMU herbicide (FIG. 12D). 0012 FIG. 2 is a group of digital AFM images of gold 0024 FIG. 13 is a graph illustrating the effect of light electrodes modified with (a) MWNT without thylakoid (b) intensity on thylakoid-MWNT composites. High light inten thylakoid-MWNT composite and (c) thylakoid without sity was turned on at 60s, medium at 540s and low light at MWNT. The topography, amplitude, and phase images cor 92OS. respond to left, middle and right columns respectively. Thy (0025 FIGS. 14A-14B illustrate photocurrent analysis of lakoids are marked by the arrows in the images. thylakoid-MWNT modified gold electrode under: constant 0013 FIGS. 3A-3B are a group of SEM images of bare light (FIG. 14A) and constant dark (FIG. B) conditions, as electrode modified with (3A) thylakoids, (3B) MWNT and compared to light/dark cycle. (3C) thylakoids-MWNT composite. Thylakoids are shown (0026 FIGS. 15A-15B illustrate a comparison of immobi by the arrows. lized thylakoids (1x) with various quantities (20x, 100x, 0014 FIGS. 4A-4B are graphs of cyclic voltammograms 200x) of suspended thylakoids in solution. FIG. 15A is a (CV) of a thylakoid-MWNT composite modified electrode. graph of photocurrent response ( represents light condition FIG. 4A illustrates the CV in the presence and absence of 1.5 and represents dark condition), FIG. 15B illustrates cyclic mM mediator; inset graph shows capacitance Subtracted Vol voltammograms under light. Note: 1x corresponds to 0.014 tammogram. FIG. 4B illustrates the CV under light and dark Ill9 it. conditions with 1.5 mM mediator; inset graph shows the 0027 FIG. 16A illustrates cyclic voltammograms of thy background Subtracted Voltammograms. 1 peak 2,pea and lakoid-MWNT composites with and without exposure to 3 represent the redox reactions of cyt b6f ferricyanide DCMU herbicide, and FIG. 16B illustrates background sub mediator, and plastocyanin, respectively. tracted cyclic Voltammograms showing the retention of redox 0015 FIG. 5A is a graph of pen circuit potentials of peaks of cyt-bf (0V) and plastocyanin (0.2V). unmodified (control) and thylakoid modified MWNT elec 0028 FIG. 17 is a graph illustrating a comparison of the trodes in the presence of 1.5 mM Fe(CN). FIG. 5B photo-current responses of unexposed and paraquat exposed illustrates a graph of photo-current responses of unmodified thylakoid-MWNT composites. and represents light on (control) and thylakoid modified MWNT electrodes at a fixed and light off conditions, respectively. potential of 0.2V in the presence of 1.5 mM Fe(CN). 0029 FIG. 18 is agroup of scanning Electron Microscopic 0016 FIG. 5C illustrates Nyquist plots (Z vs. -Z") for images: (a) carbon paper, (b) carbon paper with MWCNT thylakoid-MWNT composites under light and dark condi (CP-MWCNT), (c) Anabaena variabilis immobilized on CP tions. Inset in FIG. 5C shows the equivalent circuit model MWCNT and (d) Nostoc sp. immobilized on CP-MWCNT. used to fit the Nyquist data. In FIGS. 5A and 5B, and 0030 FIG. 19A is a graph illustrating measurement of represent light on and light off conditions, respectively. open circuit potential comparing bare and PS bacteria-modi 0017 FIG. 6 is a graph illustrating a comparison of the fied CP-MWCNT electrodes, and the graph in FIG. 19B photo-current responses of unexposed and DCMU exposed illustrates photocurrent density ofbare and PS bacteria modi thylakoid-MWNT composites. and represent light on and fied CP-MWCNT. light off conditions, respectively. 0031 FIG. 20 is a graph illustrating dependency of pho 0018 FIG. 7A illustrates a schematic representation of an embodiments of a photo-electrochemical cell of the present tocurrent generation on Nostoc sp. loading on the disclosure containing thylakoid-MWNT based photo-anode CP-MWCNT electrode. and laccase-MWNT based biocathode. FIG. 7B is a graph of 0032 FIG. 21 is a graph illustrating dependency of pho the steady state polarization and power density curves of the tocurrent generation on AV loading on the CP-MWCNT elec photo-electrochemical cell. A digital image of the simple trode. photo-electrochemical cell setup is shown in the inset. 0033 FIG. 22 is a graph illustrating photocurrent genera 0019 FIG. 8 illustrates a UV-Vis spectrum of suspended tion in Nostoc sp. illuminated with white light of varying thylakoid membranes used to calculate chlorophyll concen intensities. tration. 0034 FIG. 23 is a graph illustrating the stability of pho 0020 FIGS. 9A-D are graphs illustrating photocurrent tocurrent generation in Nostoc sp. under continuous light and analysis of thylakoid-MWNT composites for the optimiza dark and alternate light/dark phases. tion of (9A) thylakoid immobilization time, (9B) chlorophyll 0035 FIGS. 24A and 24B are graphs, with FIG. 24A loading, (9C) mediator concentration and (9D) applied poten illustrating absorption spectrum of Nostoc and A. variablis in tial. represents light condition and represents dark con the visible light spectrum, while FIG. 24B illustrates the dition. An applied potential of 0.2V. 1 hr thylakoid immobi measurement of photocurrent of Nostoc sp. at different char lization time, and 1.5 mM mediator concentration were the acteristic wavelengths. most favorable conditions for this technique. 0036 FIG.25 illustrates the photosynthetic electron trans 0021 FIG. 10 illustrates cyclic voltammograms of thyla port chain and blocking sites of various inhibitors. koid-MWNT composites in the presence and absence of 10 0037 FIG. 26A is a graph illustrating the effect of various mM KCN as plastocyanic inhibitor. inhibitors of photosynthetic electron transport chain on pho 0022 FIG. 11 is a graph illustrating photocurrent tocurrent generation in Nostoc sp., and FIG. 26B is a bar graph responses of 1.5 mM 2,6-dichloro-p-benzoquinone and 50 showing the percentage inhibition of each inhibitor (DCMU, mM 1,4-benzoquinone mediators on MWNT electrodes in DBMIB, and KCM) the absence of thylakoids. The photocurrent response offer 0038 FIG. 27A illustrates the effect of varying concentra ricyanide mediator is also shown for comparison. tion of DCMU on photocurrent generation in Nostoc sp. FIG. US 2014/003 8065 A1 Feb. 6, 2014
27B is a bar graph illustrating the decrease in percentage without departing from the scope or spirit of the present photocurrent as a function of concentration of DCMU. disclosure. Any recited method can be carried out in the order 0039 FIG. 28 illustrates the effect of varying concentra of events recited or in any other order that is logically pos tion of DBMIB on photocurrent generation in Nostoc sp. sible. 0040 FIG. 29A illustrates the effect of varying concentra 0047 Embodiments of the present disclosure will employ, tion of KCN on photocurrent generation in Nostoc sp. FIG. unless otherwise indicated, techniques of molecular biology, 29B is a bar graph illustrating the decrease in percentage microbiology, organic chemistry, biochemistry, genetics, photocurrent as a function of concentration of KCN. botany, electrochemistry and the like, which are within the skill of the art. Such techniques are explained fully in the DESCRIPTION literature. 0041. The details of some embodiments of the present 0048. It must be noted that, as used in the specification and disclosure are set forth in the description below. Other fea the appended claims, the singular forms “a,” “an and “the tures, objects, and advantages of the present disclosure will be include plural referents unless the context clearly dictates apparent to one of skill in the art upon examination of the otherwise. Thus, for example, reference to “a support' following description, drawings, examples and claims. It is includes a plurality of supports. In this specification and in the intended that all Such additional systems, methods, features, claims that follow, reference will be made to a number of and advantages be included within this description, be within terms that shall be defined to have the following meanings the scope of the present disclosure, and be protected by the unless a contrary intention is apparent. accompanying claims 0049. As used herein, the following terms have the mean 0042. Before the present disclosure is described in greater ings ascribed to them unless specified otherwise. In this dis detail, it is to be understood that this disclosure is not limited closure, “comprises.” “comprising.” “containing” and "hav to particular embodiments described, and as such may, of ing” and the like can have the meaning ascribed to them in course, vary. It is also to be understood that the terminology U.S. Patent law and can mean “includes,” “including,” and the used herein is for the purpose of describing particular like: “consisting essentially of or “consists essentially” or embodiments only, and is not intended to be limiting, since the like, when applied to methods and compositions encom the scope of the present disclosure will be limited only by the passed by the present disclosure refers to compositions like appended claims. those disclosed herein, but which may contain additional 0043. Where a range of values is provided, it is understood structural groups, composition components or method steps. that each intervening value, to the tenth of the unit of the lower Such additional structural groups, composition components limit unless the context clearly dictates otherwise, between or method steps, etc., however, do not materially affect the the upper and lower limit of that range and any other stated or basic and novel characteristic(s) of the compositions or meth intervening value in that stated range, is encompassed within ods, compared to those of the corresponding compositions or the disclosure. The upper and lower limits of these smaller methods disclosed herein. “Consisting essentially of or ranges may independently be included in the Smaller ranges “consists essentially” or the like, when applied to methods and are also encompassed within the disclosure, Subject to and compositions encompassed by the present disclosure any specifically excluded limit in the stated range. Where the have the meaning ascribed in U.S. Patent law and the term is stated range includes one or both of the limits, ranges exclud open-ended, allowing for the presence of more than that ing either or both of those included limits are also included in which is recited so long as basic or novel characteristics of the disclosure. that which is recited is not changed by the presence of more 0044) Unless defined otherwise, all technical and scien than that which is recited, but excludes prior art embodi tific terms used herein have the same meaning as commonly mentS. understood by one of ordinary skill in the art to which this 0050. Prior to describing the various embodiments, the disclosure belongs. Although any methods and materials following definitions are provided and should be used unless similar or equivalent to those described herein can also be otherwise indicated. used in the practice or testing of the present disclosure, the preferred methods and materials are now described. DEFINITIONS 0045 All publications and patents cited in this specifica tion are herein incorporated by reference as if each individual 0051. In describing and claiming the disclosed subject publication or patent were specifically and individually indi matter, the following terminology will be used in accordance cated to be incorporated by reference and are incorporated with the definitions set forth below. herein by reference to disclose and describe the methods 0052. As used herein, the term “photosynthetic com and/or materials in connection with which the publications pound includes any compound involved in the photosyn are cited. The citation of any publication is for its disclosure thetic process, e.g., the process of harnessing light energy to prior to the filing date and should not be construed as an induce a photochemical reaction to oxidize water molecules admission that the present disclosure is not entitled to ante and generate electrons. “Photosynthetic compounds” date such publication by virtue of prior disclosure. Further, includes “photosynthetic proteins’ and protein complexes, the dates of publication provided could be different from the such as, but not limited to, PSI, PSII, cyt bef, plastocyanin, actual publication dates that may need to be independently phycocyanin, and phycoerythrinas well as other non-protein, confirmed. photosynthetic molecules, such as, but not limited to, plasto 0046. As will be apparent to those of skill in the art upon quinone and carotenoids. The photosynthetic compounds of reading this disclosure, each of the individual embodiments the present disclosure may be isolated from the host organism described and illustrated herein has discrete components and and organelles in which they originate, or they may be located features which may be readily separated from or combined in a thylakoid membrane or thylakoid organelle or photosyn with the features of any of the other several embodiments thetic bacterial organism. US 2014/003 8065 A1 Feb. 6, 2014
0053 As used herein, the term “photosynthetic reaction electric current using light induced photo-electrochemical center” (PSRC) refers to one or more photosynthetic com reactions catalyzed by photosynthetic compounds. The pounds as defined above. A PSRC may include a single pho present disclosure also includes methods of generating an tosynthetic compound (e.g., PSII) or it may contain a group of electrical current using photosynthetic compounds, thylakoid photosynthetic compounds, whether isolated or working in a membranes, and/or photosynthetic bacteria or portions of cluster or entity (e.g., thylakoid membrane or photosynthetic photosynthetic bacteria to harness light energy and using organism). APSRC, as used in the present disclosure, has the direct electrochemical communication to transfer electrons ability to harness light energy to induce a photochemical generated by the photosynthetic proteins to an electrode. reaction to oxidize water molecules and generate electrons. 0061. In embodiments of the photosynthetic electro 0054 As used in the present disclosure, two materials are chemical cells of the present disclosure, the cell includes an in “electrochemical communication' when electrons gener anode composite that includes an anode (Substrate electrode) ated by a chemical reaction of one material (e.g., photosyn and a photosynthetic reaction center (catalyst) including one thetic reaction centers) can be transferred to and/or accepted or more photosynthetic compounds. The cell also includes a by the other material (e.g., nanostructured material and/or cathode or cathode composite including a cathode (substrate electrode). electrode) and at least one enzyme or metallic catalyst 0055 “Direct electron transfer” as used in the present capable of reducing oxygen or other reductant. In embodi application indicates that an electron can be transferred to an ments of the present disclosure, the anode composite har electrode (e.g., anode) from the photosynthetic reaction cen nesses light energy to oxidize water molecules and generate ter (PSRC) that catalyzed the reaction that produced the elec electrons for transfer to the cathode for reduction of oxygen. tron, as opposed to having to be transferred to the electrode by Thus, the cathode uses electrons from the anode to reduce O. a separate shuttle molecule (e.g., a redox mediator or redox which induces a potential difference between the anode and shuttle). In the present application, direct electron transfer the cathode and generates an electrical current. The methods includes the transfer of electrons generated from a photosyn and the photosynthetic electrochemical cells of the present thetic protein to the electrode through a nanostructured mate disclosure, therefore, provide a method of harnessing light rial or matrix of nanostructured materials, such as where the energy to generate an electrical current through photo-in nanostructured material couples the photosynthetic proteins, duced electrochemical reactions and direct electron transfer. thylakoid membrane and/or photosynthetic organism to the 0062. In embodiments, the anode composite includes a electrode. The presence of direct electron transfer in an elec photosynthetic reaction center (PSRC), or multiple PSRCs. trochemical cell of the present disclosure does not preclude The PSRC includes one or more photosynthetic compounds, the existence of Some electron transfer occurring through a and Such photosynthetic compounds can include proteins, mediator, it just indicates that direct electron transfer is occur pigments, protein/pigment complexes, and other non-protein ring in the cell. compounds involved in photosynthesis. In embodiments of 0056. As used herein, the term “anode composite' refers photosynthetic electrochemical cells of the present disclo to a construct that provides the anode function in a photosyn sure, the PSRC of the anode composite includes at least one thetic electrochemical cell of the present disclosure. Thus, the photosynthetic protein capable of oxidizing water molecules anode composite includes the anode as well as any other and generating electrons using light induced photo-electro materials or components coupled to the anode that provide for chemical reaction. The electrons produced by the PSRCs are the oxidizing capability of the anode (e.g., nanostructured conducted to the anode via direct electron transfer. While not matrix material, photosynthetic reaction centers, and the like, every electron produced by the PSRC will necessarily be as well as compounds or liking agents used to couple the conducted to the anode via direct electron transfer (some may anode to the other components of the anode composite). be lost, and in Some embodiments, a portion of the electrons Similarly, the term “cathode composite' refers to a construct generated may be transferred to the anode via a redox media that includes the cathode as well as other materials that pro tor), it will be understood that at least a portion of the elec vide for the reducing activity of the cathode (e.g., the cathode trons are conducted to the anode via direct electron transfer. and a compound capable of reducing O, as well as any In embodiments of the photosynthetic electrochemical cells compounds or agents used to couple the cathode to the other of the present disclosure, the PSRC includes one or more of components of the cathode composite, such as nanostruc the following photosynthetic compounds: PSII, PSI, plasto tured materials and/or any linking agents). quinone, cyt bef, plastocyanin, phycocyanin, phycoerythrin, 0057 The term “matrix of nanostructured materials', as and carotenoids. The PSRC may include a combination of the used in the present disclosure, includes a network or multi above photosynthetic compounds. In embodiments, the dimensional structure of nanoparticles capable of coupling PSRC of the electrochemical cell of the present disclosure photosynthetic proteins, a thylakoid membrane or organelle includes at least one photosynthetic protein or protein com to an electrode. plex, such as, but not limited to, PSI, PSII, cyt bef, plastocya 0058 “Redox mediator or “redox shuttle' refers to a nin, phycocyanin, and phycoerythrin. In embodiments, the compound capable of assisting in the transfer of electrons PSRC can also include non-protein photosynthetic com between a redox enzyme (e.g., a photosynthetic compound of pounds such as, but not limited to, plastoquinone and caro the present disclosure that oxidizes water and generates elec tenoids. In embodiments, the PSRC may include one or more, trons) and an electrode. two or more, three or more, or any combination of the above 0059 Having defined some of the terms herein, the vari photosynthetic compounds. In embodiments, the anode com ous embodiments of the disclosure will be described. posite may include one or more PSRCs where each PSRC may include one or more photosynthetic compounds. DESCRIPTION 0063. The photosynthetic proteins and compounds 0060 Embodiments of the present disclosure include pho included in the photosynthetic electrochemical cells of the tosynthetic electrochemical cells capable of generating an present disclosure may be isolated (e.g., removed from their US 2014/003 8065 A1 Feb. 6, 2014
natural environment, organism, organelle, membrane, etc.) or 0067. In embodiments, the nanostructured material is a they may be included in a thylakoid membrane, an in-tact matrix of nanostructured material made of a material capable thylakoid organelle, a photosynthetic organism (e.g. a photo of being coupled to and in electrochemical communication synthetic bacterium) or a photosynthetic portion of a photo with the PSRCs. In embodiments, the nanostructured mate synthetic organism (e.g., a portion of the organism that is rials include, but are not limited to, carbon based nanomate capable of photosynthesis when isolated from the source rials, metallic nanoparticles, semiconductor nanoparticles, organism). quantum dots or combinations of these materials. Some 0064. In embodiments of the photosynthetic electro embodiments of carbon based nanomaterials useful for the chemical cells of the present disclosure, the PSRCs are electrochemical cells of the present disclosure include, but included in a photosynthetic entity coupled to the anode. In are not limited to, materials such as carbon nanotubes, multi Some embodiments where the photosynthetic compounds are walled carbon nanotubes, fullerenes, carbon nanoparticles, isolated from their natural environment, they can be included graphenes, two dimensional carbon nanosheets, graphite in a synthetic photosynthetic structure (e.g., a structure made platelets, and the like. In some specific embodiments, the of nanostructured materials, such as the matrix of nanostruc matrix of nanostructured material is multi-walled carbon tured materials described in greater detail below). In other nanotubes. embodiments the photosynthetic compounds are present in a 0068. In the electrochemical cells of the present disclo natural photosynthetic entity (e.g., a thylakoid organelle, a sure, in embodiments, the PSRCs are coupled to the matrix of thylakoid membrane, a photosynthetic organism, or a portion nanostructured material in the anode by a cross-linking agent, of a photosynthetic organism). Using the proteins in a natural Such as, but not limited to, 1-pyrenebutanoic acid Succinim environment, such as a thylakoid membrane or photosyn idyl ester (PBSE) or other protein homo- or hetero-bifunc thetic bacterium, may provide certain advantages, such as tional cross-linking agent. facilitating the coordinated transfer of electrons between the 0069 FIG. 1 provides an illustration of an embodiment of various photosynthetic compounds in the membrane/organ an anode composite of the present disclosure, showing the ism. This offers various pathways for electron transfer electrode, the matrix of nanostructured materials provided as between the photosynthetic compounds in the PSRCs and the multi-walled carbon nanotubes (MWCNTs), and a thylakoid anode, rather than just a single path offered by a single iso membrane as a photosynthetic entity/PSRC including a com lated photosynthetic protein. Thus, in some embodiments it bination of photosynthetic proteins and other photosynthetic may be advantageous to utilize natural photosynthetic enti compounds in electrochemical communication with the ties, such as a portion of a thylakoid membrane, an intact anode via the matrix of MWCNTs and PBSE likers. thylakoid organelle, or photosynthetic organism, or part 0070 Although the methods and photosynthetic electro thereof in the electrochemical cells of the present disclosure. chemical cells of the present disclosure allow for direct elec 0065. As used herein, a PSRC can refer to an isolated tron transfer between the PSRCs and the anode, in some photosynthetic compound, to a grouping or cluster of photo instances it may be advantageous to include a redox mediator synthetic compounds working together, to a synthetic struc (also known as a redox shuttle) to facilitate transfer of elec ture including a cluster of photosynthetic compounds, to the trons between the photosynthetic proteins and the nanostruc photosynthetic compounds or groups of compounds within tured material/anode. In embodiments the redox mediator Such a synthetic structure, to a single photosynthetic entity may be chosen from mediators such as, but not limited to, Such as a thylakoid membrane, thylakoid organelle or photo ferricyanide, quinone-based compounds, osmium complex synthetic organism, or to a group of photosynthetic com based compounds, any other redox chemical compound and pounds within such a photosynthetic entity. Thus, a PSRC combinations of the above. refers to any single or grouping of photosynthetic compounds 0071. In embodiments of the photosynthetic electro capable of oxidizing water molecules and generating elec chemical cells of the present disclosure, the cathode includes trons using light induced photo-electrochemical reactions. at least one compound capable of reducing a reductant, Such 0066. The photosynthetic compounds (whether isolated or as, but not limited to O., ferro/ferricyanide couple, and the part of a natural or synthetic photosynthetic structure) are like. In embodiments, the photosynthetic electrochemical included in the anode composite such that the PSRCs are in cells of the present disclosure include a cathode composite electrochemical communication with the anode so that elec including a cathode and a compound or combination of com trons generated during photosynthetic reactions can be con pounds capable of reducing a reductant. Such as O. In ducted directly to the anode. In some embodiments, the embodiments, the cathode composite may also include a PSRCs are coupled to the anode by a nanostructured material. nanostructured material to facilitate electrochemical commu In Such embodiments, the anode composite also includes a nication between the cathode and the oxygen-reducing com nanostructured material in electrochemical communication pounds. The nanostructured material of the cathode compos with at least one PSRC and the anode. In embodiments where ite can be any of the nanostructured materials described above the PSRCS are isolated photosynthetic compounds or clusters in reference to the anode composite. In embodiments, the of isolated compounds, the PSRCs may be coupled to and/or compound capable of reducing O can include, but is not integrated into a nanostructured material Such that they form limited to, an enzyme or a metallic catalyst. In some embodi a synthetic photosynthetic structure, as described above, and ments the enzyme capable of reducing O. can include, but is the photosynthetic structure can be coupled directly to the not limited to, laccase, bilirubin oxidase, ascorbate oxidase, anode. In other embodiments, the PSRCs may be a natural tyrosinase, catechol oxidase, and combinations of these or photosynthetic entity, and the PSRC/entity may be coupled to other enzymes. In embodiments the enzyme capable of reduc the anode via a matrix of nanostructured material. In some ing O include a metallic catalysts such as, but not limited to embodiments, the anode can be functionalized with a nano platinum, silver, gold, cobalt, nickel, iron and combinations structured material, and the PSRC is in electrochemical com of these metals. In embodiments, the compound capable of munication with the nanostructured material of the anode. reducing ferrofferricyanide couple, can include but is not US 2014/003 8065 A1 Feb. 6, 2014
limited to, could be a metal, semiconductor or carbon or a (0077. Now having described the embodiments of the chemical capable of reducing the ferrofferricyanide couple. present disclosure, in general, the Examples, below, describe 0072. In embodiments of the present disclosure the anode Some additional embodiments of the present disclosure. and cathode can be made of any standard electrode material, While embodiments of the present disclosure are described in Such as carbon, metals, semiconductor Such as silicon, and the connection with the Examples and the corresponding text and like. One advantage to the cells of the present disclosure is figures, there is no intent to limit embodiments of the present that the use of the nanostructured materials to couple the disclosure to these descriptions. On the contrary, the intent is photosynthetic compounds or oxygen reducing compounds to cover all alternatives, modifications, and equivalents to the electrodes allows coupling of the photosynthetic com included within the spirit and scope of embodiments of the pounds to the electrode without the need for expensive pre present disclosure. cious metal electrodes (e.g., gold, silver, platinum, etc.) and without complex immobilization procedures that are incom EXAMPLES patible with other, less expensive electrode materials such as carbon. Example 1 0073. The examples below and the attached figures pro Manipulating Electron Transport Pathways in vide additional detail regarding some embodiments of the Thylakoid Composites for Photosynthetic Energy photosynthetic electrochemical cells of the present disclosure Conversion and the elements of the anode and cathode described above. 0078 Spinach thylakoids were coupled to electrodes via FIG. 7A provides an illustration of an embodiment of an multiwalled carbon nanotubes using a molecular tethering electrochemical cell of the present disclosure. chemistry. The resulting thylakoid-carbon nanotube compos 0074 The methods of the present disclosure include meth ite showed high photo electrochemical activity under illumi ods of generating an electrical current including using the nation. It is believed to be the first time multiple membrane photosynthetic electrochemical cells of the present disclosure proteins have been observed to participate in direct electron to convert light energy to electrical energy. In embodiments, transfer with the electrode, resulting in the generation of the methods of the present disclosure include using thylakoid photocurrents. Thus, it is believed that the present disclosure membrane photosynthetic proteins and compounds and/or describes the first of its kind for natural photosynthetic sys photosynthetic bacterial proteins and compounds to harness temS. light energy to oxidize a water molecule and directly transfer 007.9 The high electrochemical activity of the thylakoid electrons from the photosynthetic proteins to an anode using MWNT composites has significant implications for both pho direct electrochemical communication. In embodiments, tosynthetic energy conversion and photofuel production methods of the present disclosure also include using the elec applications. A fuel cell type photosynthetic electrochemical trons from the anode to reduce O at a cathode, thereby cell developed using thylakoid-MWNT composite anode and inducing a potential difference between the anode and the laccase cathode produced a maximum power density of 5.3 cathode and generating an electrical current. uW cm’ comparable to that of enzymatic fuel cells. The 0075 Briefly described, some methods of generating an carbon based nanostructured electrode has the potential to electrical current according to the present disclosure include serve as an excellent immobilization Support for photosyn providing an electrochemical cell that has an anode compos thetic electrochemistry based on the molecular tethering ite that includes a PSRC in electrical communication with an approach as demonstrated in the present example. anode via a nanostructured material and a cathode composite capable of reducing oxygen. The electrochemical cell is Introduction exposed to light in the presence of water so that the PSRC uses 0080 Plant photosynthesis has evolved over 2.5 billion light energy to oxidize water molecules and generate elec years to convert Solar energy into chemical energy using only trons, which are transferred to the anode via the nanostruc water, with an unmatched quantum efficiency of nearly 100% tured material, flow to the cathode, where they reduce oxy '’. In recent years, there has been an increasing interest in gen, thereby inducing a potential difference between the mimicking the natural photosynthetic process for energy con anode and the cathode to generate an electrical current. version and photo fuel (ethanol, H., etc.) production'. This 0076. In embodiments the present disclosure includes is being carried out using synthetic routes such as metal methods of generating an electrical current by providing a oxides, semiconductors or chemical catalysts for carrying out photosynthetic electrochemical cell of the present disclosure the light-driven water splitting reaction'''. Alternatively, and exposing the photosynthetic electrochemical cell to light components of the naturally occurring photosynthetic appa in the presence of water, such that the PSRCs use light energy ratus of bacteria', algae and plants' have been to oxidize a water molecule and generate electrons which are employed for bioconversion applications. For example, the transferred to the anode via the nanostructured material, and direct conversion of light into electricity based on photosyn the electrons generated at the anode flow to the cathode where thesis in an electrochemical cell has been investigated in the they are used to reduce O at a cathode, thereby inducing a past''''', using natural systems such as thylakoids, chloro potential difference between the anode and the cathode and phyll molecules, photosynthetic reaction centers''''', and generating an electrical current. The photosynthetic electro even whole cells such as cyanobacteria’’’. Besides these chemical cells used in the methods of the present disclosure representative attempts, the low electron transfer efficiency of can include an anode composite having photosynthetic reac the photosynthetic machinery to the electrodes still plague the tions centers in electrical communication with an anode via a power output performance of these systems. Isolated photo nanostructured material as described above and include a synthetic components systems possess Some advantages over cathode or cathode composite capable of reducing O or other whole cells, such not needing nutrients for Sustenance, and reductant as described above. not having competition between respiration and photosynthe US 2014/003 8065 A1 Feb. 6, 2014
sis in sharing the electron transfer pathways. However when MWNT modified electrode and the associated electron trans isolated plant photosynthetic systems have been used on elec port pathways are shown conceptually in FIG. 1. trodes they have suffered from low efficiencies due biomol ecule stability, improper immobilization, lack of electrical EXPERIMENTAL communication, etc'. For light energy harvesting applica tions, it is thermodynamically advantageous to collect elec Materials trons directly from the molecules at high-energy states along I0083) Thylakoids were extracted from fresh organic spin the photosynthetic electron transport pathway, Such as an ach obtained from local market. MWNT, 10 nm diameter and excited photosystem II (PSII)'''. Moreover, for direct 1-2 um length (DropSens, Spain) was used as the immobili light-electricity conversion applications, it is generally pref Zation Support for the thylakoids. 1-pyrenebutanoic acid Suc erable to use a higher order plant based system that uses only cinimidyl ester (PBSE) (Anaspec) was used as the molecular water as the electron donor such as PSII, rather than isolated tethering agent to attach thylakoids on MWNT. Potassium PSI complexes, which require an alternate electron donor. For ferricyanide, redox mediator, and N,N-dimethyl formamide this purpose, attempts have been made to immobilize PSII (DMF), solvent used for reagent preparation, were purchased reaction centers on to the electrode using cytochromes'' or from Acros Organics. 3-(3,4-dichlorophenyl)-1,1-dimethy nickel-nitrilotriacetic acid as cross-linkers or through lurea (DCMU) was purchased from Tokyo Chemical Indus someterminal electronacceptors such as Co' complexes’. try. Laccase from Trametes versicolor (Sigma) was the All these methods use precious metals (e.g., gold) as the enzyme used on the cathode. Potassium cyanide (KCN) was immobilization Support and use expensive immobilization purchased from Fisher Scientific. Paraquat/Diguat was pur procedures that cannot be extended to other electrode mate chased from Ultra Scientific. Tricine (OmniPur), sorbitol rials (e.g., carbon). Accordingly, the precious metal based (EMD Chemicals Inc), ethylenediaminetetraacetic acid electrochemistry carries less practical value for energy con (EDTA) (VWR), and potassium hydroxide (Mallinckrodt version applications. Baker) were used for preparing buffer solutions. Phosphate 0081. On the other hand, photosynthetic organelles or buffer for electrochemical testing was prepared using membranes also possess advantages over isolated reaction monobasic and dibasic potassium phosphates (VWR). All center complexes for electrochemical applications. Some buffers were prepared using nanopure distilled water such advantages include: high individual protein stability’, (ddHO). Electrolyte (buffer) solutions were purged for 30 the ability to use simpler immobilization procedures, and the min with N to remove any dissolved O. presence of multiple electron transfer routes. For example, if thylakoid membranes are used in the place of isolated PSII Methods complex, then the electron transfer from an oxygen evolving 0084 Thylakoids were isolated from Spinacia oleracea complex (OEC) site to the electrode can be achieved via (spinach) leaves based on the procedure given in literature (39) plastoquinone, cytochrome (cyt) bef, plastocyanin, ferre using a refrigerated centrifuge (Beckman Coulter Avanti J-E). doxin, PSI, etc. in addition to a direct transfer from PSII. The procedure is known to produce a mixture of both intact Moreover retention of their natural partners results in organelles and broken thylakoid membranes. During isola enhanced stability of the individual proteins in thylakoids in tion the chlorophyll concentration was determined to be comparison to their isolated counterparts. Therefore using between 2.5 and 3.0 mg ml (average of 2.8 mg mL) via thylakoids as photo-biocatalysts or otherwise complexing UV-Vis absorbance measurements using a spectrophotom photosynthetic proteins within a photosynthetic structure eter (UV-Vis) (Cary Varian 50 Bio, Sparta, N.J.) using the offers the potential for high photo-electrochemical activity as formula given in the FIG.8. The oxygen evolution activity of well as high stability for both energy conversion and fuel isolated thylakoids was measured in oxygen deprived tricine production applications. buffer pH 7.8 with a standard Clark O. electrode. The isolated 0082. The present example demonstrates the photo-elec thylakoids were in the form of a pellet and was stored in the trochemical activity of spinachthylakoids immobilized on to dark at -80° C. The pellets were re-suspended in the buffer multi-walled carbon nanotube (MWNT) modified electrodes. when needed for electrode preparation. For control experi By employing a carbon-based material, the necessity for ments, the DCMU exposed thylakoids were prepared by sus expensive precious metal-based catalyst Supports to immobi pending thylakoids in 0.1 mMDCMU solution and incubated lize photosynthetic machinery onto electrode Surfaces was in ice bath for 30 min. Similarly in another control experi eliminated. The molecular tethering approach described in ment, 250 ug mL' solution of paraquat/diquat solution was the present example by using nanostructured materials to used to suspend thylakoids and incubated in ice bath for 30 couple the photosynthetic structure to the electrode helps 1. establish multiple attachments between the thylakoid mem I0085 Slurries of MWNT were prepared by dispersing 1 branes and the electrode surface. Moreover, the present mg mL of MWNTs in 10 mM DMF by 10 min ultra soni example demonstrates that by using the entire membrane cation using an ultrasonic homogenizer (Omni International) instead of isolated photosystem complexes, manipulation of at the power output of 20 watts. The dispersion was sonicated the electron transfer pathways to achieve high electron trans again for 1 h in a bathsonic cleaner (XP-Pro). The obtained fer flux for photo current generation was possible. This MWNT dispersion was used as it is for electrode modifica example also demonstrates direct light to energy conversion tions. with water as the only input using a photosynthetic fuel cell I0086 A molecular tethering approach was used to immo composed of athylakoid based anode and laccase based cath bilize thylakoid membranes on the carbon nanotube matrix ode, first of its kind employing a plant photosynthetic mem using PBSE as the linker (e.g., tethering agent), which has brane and an enzymatic cathode operating at neutral pH. A been demonstrated to produce excellent bio-electrochemical schematic of the thylakoid membranes immobilized on characteristics'"'. In this method, first the electrodes (0.02 US 2014/003 8065 A1 Feb. 6, 2014 cm) were polished with 0.05 um alumina slurry. The pol parameters optimized were thylakoid immobilization time ished electrode was rinsed and ultrasonicated in ddHO for 8 (FIG.9A) and thylakoid concentration in the immobilization min. Then the electrode was modified with 4 uL of MWNT mixture (FIG.9B), mediator concentration (FIG.9C), and the dispersion and later dried at 70° C. After drying, a desired anode polarization potential (FIG. 9D). The results showed volume of 10 uM PBSE was drop casted on the MWNT the photocurrent was directly proportional to thylakoid con modified electrode and incubated for 15 min in an ice bath. centration; however, only a maximum of 0.44 ug cm of The resulting modified electrode was washed first with DMF thylakoids could be immobilized due to the limited geometric to remove the loosely bounded PBSE and then with tricine size of the electrodes being used in the experiment. Similarly, buffer to neutralize the pH of the electrode surface. Finally, 5 it was noticed that incubation durations (for immobilization LL of thylakoid Suspended solution (corresponds to 0.44 ug of thylakoids on MWNT matrix) beyond 1 h did not result in cm chlorophyll loading) was drop casted on the electrode a significant increase in photocurrents. The photocurrent was surface and incubated for 1 h in the dark in an ice bath. The also proportional to the mediator concentration, but the per modified electrode was then washed with tricine buffer prior cent decrease of photocurrent per duty cycle varied. The to experimentation. optimized mediator concentration obtained was 1.5 mM, where the photocurrent was 0.9 LA for the first cycle while Testing still maintaining stability through multiple cycles. The anode I0087 Bare or thylakoid modified MWNT was used as the potential was also optimized at 0.2 V to observe noticeable working electrode in a 3-electrode electrochemical cell setup photo-activity. with a platinum wire counter electrode (Alfa Aesar) and a silver-silver chloride (Ag/AgCl) reference electrode (CH Results and Discussion Instruments). All electrochemical experiments were con ducted at 25+2° C. using 0.1 M tricine buffer pH 7.8 as the Physical Characterization electrolyte. Electrochemical tests were performed both in the (0090 Tapping mode AFM and SEM were used to study presence and absence of Fe(CN) as a redox mediator. the morphology of thylakoid-modified MWNT electrodes. The operating conditions for electrochemical tests were cho The AFM topography, amplitude and phase images of the sen based on a series of optimization tests for thylakoid load unmodified MWNT (control) and thylakoid-modified ing, immobilization duration, mediator concentration and MWNT electrodes are shown in FIG. 2. The unmodified anode potential over a reasonable range, the results of which control electrode with the bare MWNT matrix shows clear are given in FIGS. 9A-9B. The O, evolution activity during and identifiable nanotubes as seen in FIG. 2A. The thylakoid experiments was monitored via a Clark-type O electrode modified-MWNT composite matrix shown in FIG. 2B has a (VWR Symphony Dissolved Oxygen Probe). Cyclic voltam similar morphology as that of the control, but with distinct metry (CV), current vs. time (i-t curve, steady state current) thylakoids on the surface. Due to the blanketing effect of and electrochemical impedance spectroscopy studies were biological structures, the underlying MWNT fibrils in the conducted using CHI-920c model potentiostat (CH Instru composite are not obvious in the topography image, but are ments). A Dolan-Jenner Industries Fiber-Lite model 190 evident in amplitude and phase images in FIG. 2B. The pres lamp was used for light illumination with high intensity ence of thylakoids is shown by the lighter extruding region in setting of 80 mW cm. Surface morphology of the immobi the composite (FIG. 2B), sized approximately 3 um+0.5 in lized thylakoids was studied using a scanning electron micro length which agrees with the typical size of a thylakoid unit scope (SEM) (FEI Inspect F FEG-SEM), atomic force micro ''. A thylakoid modified electrode surface in the absence of scope (AFM) (Veeco Multimode Nanoscope). Absorption MWNT is also shown in FIG. 2C for comparison and verifi spectra were obtained using Genesys 10S UV-Vis spectro cation. The surface roughness of the thylakoid matrix is 9 photometer (Thermo Scientific). The photosynthetic fuel cell nm,0.5, which is close to the roughness of a monolayer. The was constructed usingthylakoid-MWNT composite modified roughness values are similar for both thylakoids (FIG. 2C) anode and laccase-MWNT composite modified cathode. The and thylakoid-MWNT composite matrices (FIG. 2B). The electrodes were held inside a glass vial containing 0.1 M Surface coverage of active membrane proteins of thylakoids phosphate buffer solution (pH 6.8) as electrolyte. No oxygen on the electrode surface is discussed in section 3.3.1. The was bubbled during the experiment and the oxygen available morphology studied using SEM also show the length of thy in the electrolyte was reduced to water at the cathode. lakoids to be 3 um+1 as shown in FIG. 3. This is in good Chlorophyll Concentration Measurements agreement with the AFM observations. 0088. The chlorophyll concentration (CCh) was calculated Electron Transfer Pathways in Thylakoids by using the data from UV-V is spectrum into the equation (E1). 0091. The thylakoid membrane consists of several integral membrane proteins that could partake in electron transfer to the electrode. As schematically depicted in FIG. 1, electrons mg 8.02x A663 + 20.2 x A645 (E1) generated as a result of the photo-induced water oxidation cal mL)) 10 -> reaction at the OEC site of PSII complex in thylakoids could be conducted to the electrode in three possible routes as indicated by arrows in FIG.1. The arrows (a) indicate the first electron transfer pathway (ETP1): Optimization of Composite Composition PSII->plastoquinone->cyt bof >MWNT->electrode. ETP1 0089. The plots of current versus time at fixed anode is possible if the cyt bof is adsorbed or molecularly tethered to potentials were used as a guiding tool for optimizing the MWNTsurface and its redox site placed closely to MWNT to thylakoid-MWNT composite electrode (FIGS. 9A-D). The enable direct electron transfer. The second possible electron US 2014/003 8065 A1 Feb. 6, 2014
transfer pathway (ETP2) is depicted by arrows (b): redox peaks as observed in FIG. 4A. To confirm that the peaks PSII->plastoquinone-scyt at 0.2 V can be attributed to plastocyanin redox activity, a b6f splastocyanin->MWNT->electrode. Here the electron separate plastocyanin-inhibition experiment was conducted gets routed to plastocyanin before reaching the MWNT to study the effect of inhibition on this redox peak. For this matrix. Since plastocyanin may freely diffuse between the purpose KCN (10 mM) was added as an inhibitor to the stromal and lumenal sides of a ruptured thylakoid membrane thylakoid solution prior to immobilization. KCN inhibits the (as in the case of our experiments) it is likely to participate in plastocyanin activity in photosynthesis by blocking its Cul/II the electron conduction as well. Both ETP1 and ETP2 serve active site at high concentrations (>10 mM) and high pH as direct electron transfer routes for electrochemical charge values (>7.5)'''. The voltammograms of thylakoid modified transfer. Upon the addition of a mediator such as Fe(CN) MWNT electrode with KCN inhibitor showed a dramatic '', a third pathway as indicated by arrows (c) is also pos 73% reduction in the oxidation peak at 0.2 V as shown in sible for electron conduction to the electrode (ETP3): supplementary FIG. 10. This supports the conclusion that the PSII->plastoquinone-scyt bof?plastocyanin->Fe(CN), redox response in the 0.19 V-0.2 V range was that of plasto 4.-->MWNT->electrode. In this case, ferricyanide mediates cyanin in thylakoid modified electrodes. This is one of the few the electron transfer from multiple membrane proteins in the reports that demonstrate a direct electrochemistry for thyla thylakoid to the MWNT electrode. Therefore the lack of koid-based electrodes. electrical connectivity between those membrane proteins and electrode is not of a concern. Mediated Electron Transport 0092. In addition to the three ETPs discussed above, other 0095. In a separate set of experiments, 1.5 mM Fe(CN), routes for electrochemical communication between thyla ' couple was added to the electrolyte to assist electron koids and MWNT electrode are possible. For example, a transfer from thylakoid membrane to the MWNT electrode. direct electron transfer from plastoquinone site of PSII to Ferricyanide is a suitable choice because of its minimal MWNT is possible if the stromal side of PSII complex is photo-activity'', compared to benzoquinone mediators orientated towards the electrode. However the present experi used elsewhere’, as confirmed in separate studies (see FIG. mental results (discussed in the following paragraphs) indi 11 for relevant data). As shown in FIG. 4A, in the presence of cated no significant contribution from any additional routes to Fe(CN), a single dominant oxidation peak (2peak at EO the electrochemical charge transfer and hence were not 0.16V) was observed masking the redox responses of both cyt depicted in FIG. 1 or explored in detail. Also, since the pri b6f and plastocyanin observed. This confirms the existence of mary focus of this example is on the electrons generated at mediated electron transport pathway (ETP3) as suggested in PSII complex by water splitting reaction, other ETPs origi FIG.1. This also indicates that the electron flux due to medi nating from PSI or from electrolyte impurities' are not ated transport was much higher than that of direct transport discussed here. through the membrane bound proteins. The O evolution due to photo-induced water splitting in thylakoid modified Redox Electrochemistry of Immobilized Thylakoid MWNT electrodes upon illumination is also evident in the Membranes voltammograms of FIG. 4B. The oxygen reduction and fer 0093. Direct Electron Transport ricyanide oxidation were observed at E=-0.4V and E, 0.3 0094. Cyclic voltammetry was used to study redox activity V vs. Ag/AgCl respectively in FIG. 4B. Upon illumination the of the unmodified and thylakoid-MWNT composite modified peak currents for their reactions increased by 1.0 (I) and 0.2 electrodes and to verify the existence of the ETPs discussed (I) LLA respectively indicative of photo-catalysis by immo above in FIG.1. The electrodes were cycled between -0.7 to bilized thylakoids. The rate of oxygen evolution from the 0.5 V vs. Ag/AgCl at a scan rate of 0.02 V s'. FIG. 4A photo-induced water oxidation was measured to be 253 umol compares the cyclic voltammograms of thylakoid-MWNT O2 mg chl'h' (using a Clark electrode) indicating high composites in the presence and absence of Fe(CN) as photo-activity of the reactions centers complexes in thyla mediator. The formal potential (E') values for peaks 1peak and koids. The results suggest that the electron flux to electrode 3 as observed in our results were at -0.035 and -0.2V (see could be greatly enhanced by Fe(CN), which acts as FIG. 4A), which fell closely with that of the redox potentials mediator for electrochemical charge transfer between thyla of cyt bf (Fe') and plastocyanin (Cu'), respectively, koid and MWNT electrode. To ensure that the mediator did when the pH difference was accounted". No redox peak not interfere with light-absorbance activity of thylakoids, directly attributable to plastoquinone was observed in our individual absorption spectra were obtained for both thyla experiments, which is located inside the thylakoid lipid koids (in 80% acetone solution) and Fe(CN) couple bilayer. The surface coverage of the cyt bef and plastocyanin using UV-Vis spectroscopy. As shown in FIG. 12A, the was 1.2x10 and 0.6x10 mol cm' respectively. These absorption spectrum for Fe(CN)' mediator did not values were calculated using the equation T=Q/nFA, where T show any absorbance at 673 nm, the wavelength at which is Surface coverage, Q is the charge measured from the cyclic chlorophyll-a absorbs light in PSII. Also the presence of Voltammogram's cathodic peak, n is the number of electrons, mediator did not hinder the light absorbance of chlorophyll-a F is the Faraday constant, and A is the geometric area of the molecule in our thylakoid-MWNT composites (see FIG. 12A electrode. Since thylakoids may exist as broken membrane for relevant data). particles instead of intact organelles in our samples, the obser vance of redox peaks for alumen-side soluble protein Such as Photo-Electrochemical Activity of Thylakoid-MWNT plastocyanin is not surprising. The cyt bef and plastocyanin Composites redox centers are much smaller than other reaction center 0096. The photo-electrochemical activity of thylakoid complexes such as PSII and PSI. Therefore, naturally the modified MWNT electrodes were evaluated using open cir MWNTs have easier access to cyt bef and plastocyanin for a cuit potential (OCP), potentiostatic polarization, and AC direct electrochemical charge transfer resulting in distinct impedance measurements. US 2014/003 8065 A1 Feb. 6, 2014
Open Circuit Potentials absorbance of chlorophyll in thylakoids in the 660-675 nm. range (as shown in the absorption spectrum in Supplementary 0097 FIG. 5A compares the open circuit potentials FIG. 12A). In order to understand if immobilized thylakoids (OCPs) of unmodified and thylakoid modified MWNT elec have significant advantages over freestanding Suspended thy trodes in the presence of mediator. Within 100 seconds after lakoids in Solution, separate polarization experiments were the addition of mediatorunder dark condition, the OCP of the performed using thylakoids Suspended in the electrolyte unmodified and thylakoid modified MWNT electrodes stabi without immobilization on MWNT electrodes. For such elec lized at 0.23 and 0.19 V. respectively. The slightly lower OCP trodes the photo-electrochemical activity was drastically for thylakoid-MWNT electrode could be dictated by the reduced (as shown in FIGS. 15A and 15B), indicating that the mixed potential caused by a variety of thylakoid membrane composite based immobilization methods are best suited to proteins whose individual redox potentials range from -1.3 to establish high photo-electrochemical activity in natural sys +1.0 V vs. SHE'. Upon changing the illumination conditions temS. between light and dark over several light on-light off cycles, 0100. The decrease in the amplitude of photo-currents a clear variation in the open circuit potential was observed, overcontinuous lighton-off cycles observed in FIG.5B could indicative of photo-electrochemical activity for the thyla be due to a combination of two effects: (i) transience in koid-MWNT composite electrode. The variation was as high mediator diffusion between thylakoid and electrode before as 90 mV during the first on-off cycle, which eventually the attainment of a steady state; (ii) photo-damage of proteins Subsided during Subsequent cycles consistent with the attain under extended light exposure'. The contribution of the ment of dynamic equilibrium over time. No Such variation in first effect was studied and confirmed in a separate set of OCP between light and dark was observed for the unmodified experiments where the thylakoid-MWNT composite elec MWNT electrode (control) in FIG. 5A. trodes were tested under different duty cycles (different ratios of Time/Time) as well as under continuous light on and Potentiostatic Polarization continuous light off (dark) conditions (see Supplementary 0098. The photo-electrochemical activity of the thyla FIGS. 14A-14B for relevant data). The second effect namely koid-MWNT composite was evaluated at constant anode photo-damage (photoinhibition) occurs under continuous potential of 0.2V and the variation of anode current with time and extended illumination conditions’ and is an inherent was evaluated during light on-light off cycles. The photocur property of natural systems. In plants, natural biological rent densities ranged from 30 to 70 LLA cm during initial mechanisms repair the photo-damaged proteins. cycles eventually attaining steady state within s400s in the range of 23 to 38 LA cm that lasted for 1 week. (FIG. 13). AC Impedance Table 1 compares the photo-current densities observed in this 0101 AC impedance studies were also carried out on thy example with that of other photosynthetic systems reported in lakoid-MWNT composites under light and dark conditions in the literature and shows that the anodic photocurrents the presence of mediator to understand the influence of indi observed here are about two orders of magnitude larger than vidual resistances on photocurrent generation. The Nyquist the highest values reported in the literature for natural plant plots (-Z" vs. Z') of the impedance data and the equivalent systems, either PSII based energy conversion' or PSI electrical circuit (to which the data was fitted for parametric based bio-hydrogen production'. analysis) are given in FIG. 5C. R. represent electrolyte resis tance, R is charge transfer resistance, CPE is a constant TABLE 1. phase element between the electrolyte solution and the modi Comparison of various photosynthetic fied electrode, and lastly Z represents the Warburg imped anode's photocurrent density. ance due to the diffusion of mediator. The shape of the Nyquist profiles shows a clear difference in the impedance Electrode material Photocurrent area Reference between light and dark condition, with the composites show Au-MWNT-Thylakoid 68 LA cm This work ing lower impedance for charge transfer under the illuminated Au-poly(mercapto-p- 3.1 |IA cm 52 condition. The fitted values for R, were 87 and 317 kS2 under benzoquinone) light and dark, respectively. The results indicate an enhanced Au nanoparticles-PSII 2.4 LA cm 53 Au-Thylakoid 0.2 LA cm 60 kinetic activity due to lowered charge-transfer impedance for All-PSII 0.1 |IA cm S4 electrochemical charge transfer in thylakoid-MWNT com Au-Thylakoid 1.1 LA cm 61 posites under illumination. Though a Warburg element was All SAM-RC-RBS 0.2 LA 34 included in the equivalent circuit to ensure completeness in Carbon coated Au- 0.05 IA 62 the representation of the system, the diffusion limitations were not observed to play a major role in this kinetic limited SAM-self assembled monolayer;RC reaction center; RBS-Rhodobactersphaeroides system, as can be noticed in the shape of the Nyquist plots in Photocurrent density was calculated using the photocurrent and the active surface area FIG. 5C, where there was no 45° Warburg slope. At the same provided in the respective literature time, an equivalent circuit without the Warburg component 0099. The thylakoid-MWNT composite was also found to did not result in the perfect fit of the equivalent circuit data be very responsive to the light intensity (see FIG. 13 for light indicating that Warburg and kinetic impedances (R) were intensity data). No significant photo-electrochemical activity expressed with similar time constants. was noticed for the unmodified MWNT electrode without 0102) An impedance observation in the absence of media thylakoids or for thylakoids physically adsorbed onto the tor also showed high charge transfer impedance indicating MWNT without the tethering cross-linker. The small photo this system was not limited by the mediator diffusion. Sepa currents seen in the case of unmodified MWNT in FIG. 4B, rate measurements performed on thylakoid modified elec was primarily due to the light absorbance of Fe(CN) in trodes with and without MWNT platform indicated lower the 410-420 nm range which did not interfere with the light ohmic resistance (after eliminating the Solution impedance US 2014/003 8065 A1 Feb. 6, 2014
contribution) for the MWNT electrodes. This confirmed the anode and bilirubin oxidase cathode'’. It is worthy to point existence of high electronic conductivity for the thylakoid out that our system was not optimized for power density in MWNT composite electrodes. any way. Rather this work was a simple demonstration of Role of PSII Vs. PSI in Photocurrent Generation power generation using thylakoid membrane and laccase 0103) In order to confirm that the light-induced water cathode. Further enhancements in current and power densi splitting reaction is the electron source for the observed pho ties can be achieved by reducing the anode-cathode separa tocurrents in our composite electrodes, two control experi tion distance (to lower ohmic impedance), increasing the ments were performed. The first control experiment was loading of thylakoids and laccase onto the electrodes (to aimed at studying the effect of blocking the PSII reaction enhance electro-kinetics) and by managing the mediator dif center complex and the second was aimed at blocking the PSI fusion (to minimize mass transfer limitation) for achieving reaction center complex from participating in the photosyn high performance. thetic electron transport. For inhibiting PSII activity, 3-(3,4- dichlorophenyl)-1,1-dimethylurea (DCMU) was added to the Inhibition of Plastocyanin by KCN. thylakoid solution prior to immobilization. DCMU is a her 0105. The thylakoid-MWNT composites prepared using bicide that specifically blocks the Q->Q, site in PSII com KCN in the immobilization mixture exhibited a significant plex, severing the electron transport from PSII to the subse reduction the plastocyanin activity by up to 73%. Although quent proteins in the pathway. As shown in FIG. 6, KCN only inhibits plastocyanin, the redox activity of cyt bef potentiostatic tests at 0.2V on thylakoid-MWNT composites (peak at OV) was also reduced by 23%. This could be due to showed that when DCMU was used, the thylakoid activity the lack of an electronacceptor for cyt bef when plastocyanin was severely inhibited thereby drastically reducing the pho was inhibited), which may result in an excited cyt bef (elec tocurrents during the light on-off tests. This confirms that the tron rich) that reacts with oxygen to form peroxides that electrons originate from PSII. Interestingly the cyclic volta degrade the cyt bef activity over time as suggested by Fuerst mmograms thylakoid-MWNT composites (FIGS. 19A-19B) et. al. (E. P. Fuerst and M. A. Norman, Interaction of herbi showed retention of redox electro-activity by both cyt bef and cides with photosynthetic electron transport. Weed Science plastocyanin even when PSII was inhibited by DCMU. This suggests that blocking the PSII complex with DCMU did not 39, 458-464 (1991)). significantly affect the redox activities of the other thylakoid Photoactivity of Mediators: membrane proteins individually, an indication that the direct electron properties of thylakoid can be utilized for electro 0106 Constant potential measurements on unmodified chemical charge transfer as Suggested above. The inhibition MWNT electrodes in the presence of mediators at 0.2Vunder of PSI (as shown in supplementary FIG. 17) reveals that the light on-off conditions showed that the ferricyanide Fe(CN) presence of the PSI inhibitor did not significantly reduce the al' redox couple exhibited less photo-response than the photo-electrochemical response of the thylakoid-MWNT benzoquinone complexes used by others in the literature. composites. The results reiterate that water oxidation at PSII Therefore the observed photocurrent activity in our thyla was the major source for photocurrents in the thylakoid com koid-MWNT composite electrodes can be directly attributed posites and contribution of PSI complex to photocurrent gen to thylakoids and not the Fe(CN)' mediator. eration was insignificant in the present study. This also con firms that the major routes for electron conduction from OEC Absorption Spectra of Mediators and Inhibitors: site to the electrode were the three ETPs proposed in FIG. 1, 0107 The peak at 673 nm indicates absorbance via chlo which presumes that all the membrane integral proteins in rophyll-a. The absorption spectrum for the thylakoid-free thylakoid membrane are intact and participate in photosyn MWNT electrode did not contain the chlorophyll peak (FIG. thetic linear electron transport. 12A), indicating that the mediator does not compete chloro phyll for absorbing light in the 660-675 nm range. Also the Thylakoid-Laccase Photosynthetic Electrochemical Cell thylakoid-MWNT composite electrode in the presence of 0104. A fuel cell type electrochemical cell was con mediator showed the 664 nm chlorophyll-a peak, indicating structed using the thylakoid-MWNT composite anode and that the light absorbance of chlorophyll was not affected by laccase-MWNT composite cathode and tested in an electro the presence of mediator. Similar absorption spectra were lyte solution (PBS buffer pH 6.8). The anode oxidizes water also obtained for the composite electrodes in the presence of upon illumination with light using thylakoid-MWNT com herbicide, DCMU and the inhibitors, KCN and Paraquat/ posites as photo-biocatalysts, whereas the cathode reduces diquat. oxygen to regenerate water in the system using laccase as an enzymatic bio-electrocatalyst. The use of laccase for bio Effect of Light Intensity on Photocurrent: electrocatalytic oxygen reduction in biological fuel cells has 0108. The photo-electrochemical response of the thyla been well established''. The molecular tethering koid-MWNT composite modified electrodes varied with light approach used for thylakoid immobilization was also used for intensity as shown in FIG. 13. Initially the system was in dark. laccase immobilization on MWNT at the cathode. The open A high intensity light (80 mW cm’) was illuminated after 60 circuit potential of cell was -0.4 V. Polarization tests were sec, which resulted in the increase in measured photocurrent performed under illumination, at constant applied potentials at 0.2 V to a stable value of 2.6 LA. At time 540s, the light between 0.35 V and OV and the resulting steady state currents intensity was decreased to “medium’ setting in the lamp that were measured and shown in FIG. 7. The polarization curves resulted in the decrease in photocurrent to 1.9 LA. When light showed a maximum current density of ~70 LA/cm and a intensity was further decreased to “low” setting, the photo maximum power density of 5.3 W/cm. The power density current of the composite further decreased to 0.9 LA. The values were comparable to the ones reported for PSII electro results demonstrate very good dependency of thylakoid chemical cell using gold-poly(mercapto-p-benzoquinone) photo-response on the intensity of the incident light. In these US 2014/003 8065 A1 Feb. 6, 2014
experiments the light passes through the glass cell and buffer was noticed in the light on-off tests. However, the cyclic solution to reach the electrode, not all the intensity of light Voltammograms under light showed no loss in the redox was fallen on the electrode surface from the light source. activities of both cyt-bf and plastocyanin (FIGS. 16A-16B), indicating the direct electrochemical activity of the thylakoid Advantages of Thylakoid Immobilization: membrane redox proteins were not affected by the PSII inhi 0109 To understand if there are advantages associated bition. with immobilizing thylakoids for carrying out photo-electro chemical reactions, rather than Suspending them in the Solu Non-Involvement of PSI in Photocurrent Generation tion, separate experiments were performed. FIG. 15A reveals 0112 The non-involvement of PSI in photocurrent gen that the immobilized thylakoids exhibited a fairly stable and eration was studied by inhibiting PSI activity by paraquat/ reproducible photo-electrochemical activity over several diquat solution mixture (of the bipyridillum family). The duty cycles, whereas the thylakoids suspended in Solution solution mixture was added to thylakoids prior to immobili showed a gradual loss in the photocurrent activity with less zation. Paraquat (E -0.45 V) acts as a competitor to ferre reproducibility. Moreover, despite a high concentration of doxin (FD, E=-0.51V) for accepting the electrons from the chlorophyll in solution (up to 400 times more), the photocur F/F, site of PSI (E=-0.56V) in the photosynthetic pathway. rents of Suspended thylakoids were significantly lower than As shown in FIG. 17, the presence of the PSI inhibitor did not that of immobilized thylakoids. The electron flux of media significantly reduce the photo-electrochemical response of tors for immobilized thylakoids would be higher than for the thylakoid-MWNT composites. Therefore a portion of the Suspended thylakoids due to the proximity of thylakoid mem electron flux generated at the OEC site must have been brane proteins to the electrode, which reduces the diffusion diverted towards the electrode through Fe(CN)' media distance for mediators. It can also be noticed that although tor as depicted in FIG. 1, rather than to the PSI complex via high concentration of Suspended thylakoids increases the photocurrents, the trend is reversed at exceedingly high thy the natural pathway. lakoid concentrations due to the issues of high turbidity and CONCLUSION low light penetration in the electrolyte, a case that was care fully avoided in the above experiments. The cyclic voltam 0113. The present example demonstrated high photo-elec mograms (FIG. 15B) showed redox activities for immobi trochemical activity of immobilized thylakoid-MWNT com lized thylakoids arising from the direct interaction of surface posites for light energy harvesting application. The findings bound proteins with the electrode. For the case of thylakoids have significant implications for photosynthetic energy con Suspended in the Solution there was no such redox activity. version and photo fuel production. The composites exhibited Therefore some sort of immobilization appears to enhance direct electron transfer activity, which can be enhanced by electron transfer and high photocurrents. using a Suitable mediator. Control experiments confirmed that the light-induced water splitting reaction at the PSII complex Steady State Analysis: was the primary Source of electrons for photo-current genera 0110. When light was illuminated a large increase in tion. At least Some advantages of using thylakoid membranes anodic current was observed due to ferrocyanide oxidation at as opposed isolated photosystems lie in the self-assembly and the electrode surface. This would require a continuous ferri utilization of direct electrochemical redox activities of more cyanide reduction by thylakoid membrane proteins in the than one membrane proteins present in the thylakoid. The presence of light. Over time the current generation stabilized thylakoid-MWNT composite electrode yielded a maximum to a constant value at approximately 0.675 p.A (FIG. 14A). current density of 68 LA cm° and a steady state current This indicates that the observed decrease in photocurrent over density of 38 LLA cm, which are two orders of magnitude time during different lighton-off cycles was due to transience higher than the previously reported values in other systems in the mediator diffusion, which reaches steady state. The (Table 1). The photo-electrochemical cell delivered a maxi observed phenomenon could partly be due to the establish mum power output of 5.3 uW cm. No optimization efforts ment of steady diffusion gradients in the system. Initially to enhance the power density were attempted in this work. there was a high concentration of mediator present at the Accordingly, improvements in power densities can be real electrode-solution interface. Upon illumination, the redox ized by engineering optimization Such as, but not limited to, couple undergoes transition from ferricyanide to ferrocya designing Suitable membrane-less electrochemical cells, nide and which results in a decreased concentration of the selecting materials for electrode Substrates, developing Supe ferricyanide at the interface. This slows down electron trans rior immobilization methods etc. Additional understanding of fer to the electrode until it reaches equilibrium upon which a the electron transport pathways will help enhance direct elec steady photocurrent was observed. In the absence of light tron transfer and development of a mediator-free system to (FIG. 14B), the photocurrent was stabilized to ~1 nA. As we demonstrate direct light to electricity conversion. The bio can see from the figures, over time the currents from both inspired photosynthetic energy conversion technology using experiments reached the same steady value. This Suggests plant thylakoids demonstrated in this example offers great that the decrease in the photocurrent over time was partly due potential for green energy harvesting based on a natural pro to the transience in mediator diffusion. However a loss of cess that evolved over millions of years. photo-electrochemical activity or composite dissolution from References, each of which is incorporated herein by refer the electrode surface over time can neither be verified nor CCC. confirmed based on these experimental results. 0114 1 R. E. Blankenship, Molecular Mechanisms of Photosynthesis, Blackwell Science, Oxford, U.K., 2002. Herbicide Inhibition of Photosystem II: (0.115. 2 R. E. Blankenship, D. M. Tiede, J. Barber, G. W. 0111. Upon exposing the thylakoid to DCMU herbicide Brudvig, G. Fleming, M. Ghirardi, M. R. Gunner, W. that inhibits PSII activity, no photo-electrochemical activity Junge, D. M. Kramer, A. Melis, T. A. Moore, C. C. Moser, US 2014/003 8065 A1 Feb. 6, 2014
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(0167 54 V. Bhalla and V. ZaZubovich, Anal. Chim. Acta, possibly facilitate higher electron transfer from the electron 2011, 707, 184-190. transport pathway to the MWNT compared to AV. Electro 0168 55A. Badura, B. Esper, K. Ataka, C. Grunwald, C. chemical experiments were given in the following to verify Wöll, J. Kuhlmann, J. Heberle and M. Rögner, Photochem. this hypothesis. and Photobiol., 2006, 82, 1385-1390. 0179. In general, the value of open circuit potential (OCP) (0169 56 S. B. Powles, Ann. Rev. of Plant Physiology, is dictated by the mixed potential caused by a variety of 1984, 35, 15-44. electron transfer reactions whose individual redox potentials (0170 57 P. Sarvikas, M. Hakala-Yatkin, S. DonmeZ and E. range from -1.3 to 1.0 V vs. SHE. FIG. 19A compares the Tyystjarvi, J. Exp. Bot., 2010, 61, 4239-4247. open circuit potentials (OCP) of Nostoc sp., AV and in the (0171 58 I. Baroli and A. Melis, Planta, 1996, 198, 640 absence of bacteria (i.e., MWNT electrode). During the 646. course of the experiments, dark and light conditions (light (0172 59 L. N. M. Duysens, Biophys.J., 1972, 12, 858 on-off cycle) were varied alternatively with an interval of 300 863. S. During the lighton-off cycle, the potential of Nostoc sp. and (0173 60 J. Ahmed, W. Park and S. Kim, Bull. Korean AV modified electrodes showed stepwise variation indicating Chem. Soc., 2009, 30, 2195-2196. photo-electrochemical activity by these organisms. The 0174 61 K. B. Lam, E. A. Johnson, M. Chiao and L. Lin, potential variation between light on-off cycles was as high as J. Microelectromech. Sys., 2006, 15, 1243-1250. 100 mV for Nostoc sp. and 30 mV for AV. In the absence of (0175 62 S. A. Trammell, A. Spano, R. Price and N. Leb bacteria the electrode showed an OCP of about 0.25 V, and the edev, Biosens. Bioelect., 2006, 21, 1023-1028. OCP was constant throughout the light on-off cycle indicat ing that there was no photo-electrochemical activity on the Example 2 bare MWNT modified carbon paper (CP) electrode. 0180. The photo-activity of the photosynthetic bacteria Photosynthetic Energy Conversion using modified electrodes were evaluated at constant working elec Photosynthetic Bacteria Composites trode potential of 0.2V and the corresponding photo-current 0176). In the present example, electrochemical cells were response was measured over time during light on-off cycles. designed and tested similar to those described in Example 1, FIG. 19B shows the steady state photocurrent density above, except in place of thylakoids, photosynthetic bacterial obtained from the photosynthetic activity of both Nostoc sp. organism were complexed with carbon nanotubes to provide and AV. This increase in current density during light-on photosynthetic energy conversion. clearly indicates the transfer of electrons from the photosyn thetic bacteria to the MWNT and eventually to the electrode Materials and Methods: surface. The natural electron transport pathway of the linear photosynthetic process starts with water oxidation at the oxy 0177. Materials and methods are similar to those gen evolving complex (OEC) site of the PSII complex. The described in Example 1 above, except as noted below. Sterile electrons are then transferred from here to the PSI complex cultures of Nostoc sp. and AV were obtained from Biocon through plastoquinone, cytochrome (Cyt)bef and plastocya version Research Centre, UGA and cultured in our laboratory nin respectively. In this pathway, before the electron reaches in shake flasks using BG 11 medium under 12 hr light/dark PSI, any redox protein except PSII can transfer electrons to cycles. Once the optical density at 750 nm (OD.so) reaches the MWNT, and thus, to the electrode. The maximum photo around 1 (happens ~15 after the culture inoculation), the current density was 35 mA m for the Nostoc sp. modified culture was harvested by centrifuging at 5000 rpm for 10 min electrode, and 10 mA m° for the AV modified electrode. at room temperature and washed in phosphate buffer (0.1 M. 0181. The current vs. time plots offixed anode potentials pH 7) before used for immobilization onto the electrode. 5ul were used as a tool for optimizing the photosynthetic bacteria of multi-walled carbon nanotubes Suspension (1 mg/ml in loading on the electrode surface. The results showed that the DMF) was dropped on the carbon paper and allowed to dry. 5 photocurrent density was directly proportional to Nostoc sp. ul of the washed bacterial cells were immobilized on the top loading (FIG. 20). The maximum loading achieved on the of carbon nanotube layer, allowed to air dry. The resulting CP-MWCNT electrode during this test was 56 ug. However, bacterial cell modified carbon nanotube electrode was then higher loadings could be achieved by modifying the morphol used for the photo-electrochemical experiments. ogy and physical properties of the MWCNT coating on the CP electrode. Results and Discussion: 0182. In the case of AV (FIG. 21), a maximum loading of 0.178 The morphology of the immobilized Nostoc sp. and 128 Jug was achieved. Comparison of Nostoc sp. and AV Anabaena variabilis (AV) on the multi-walled carbon nano results reveals that Nostoc sp. Appears to possess higher tubes (MWNT) were studied by scanning electron micros photo-electrochemical activity than AV under the conditions copy (SEM). FIGS. 18A-18D shows the SEM images of tested. Since Nostoc sp. performed better among the two carbon paper, MWNT on carbon paper, Nostoc sp. on organisms studied, it was selected for detailed characteriza MWNT, and AV on MWNT. FIG. 18B shows well dispersed tion studies. MWNT uniformly deposited on the carbon paper surface 0183 The photo-electrochemical response of the Nostoc forming a mesh like matrix. The filaments of the Nostoc sp. sp. varied with incident light intensity as shown in FIG. 22. and AV can be seen in FIGS. 18C and 19D. However, careful Higher illumination resulted in higher photocurrent density, analysis of the images reveals that the sheath wrapping the in the order of 27<50<76 mW cm. The results demonstrate photosynthetic bacteria has good interaction with the MWNT the dependency of Nostoc sp. photo-response on the intensity (edges touching the MWNT in FIG. 18C). In Nostoc sp. the of the incident light. It should be noted that in the experimen interaction of sheath with the MWNT was much higher than tal set up for this example, only a small fraction of the incident at AV. This interaction of Nostoc sp. with the MWNT could light actually fell onto the electrode. This is because the US 2014/003 8065 A1 Feb. 6, 2014
electrode was kept in a hanging position inside a glass elec plete inhibition by DCMU at the Q site of PSII, or the trochemical cell containing the buffer solution and there was electron could have come from possible sources other than no attempt made to funnel the light to the electrode surface. PSII. 0188 The inhibition by DBMIB is highly dependent on 0184 The stability of current generation was studied by the concentration of the DBMIB used as illustrated in FIG. observing the steady state current change during the experi 28. At a concentration of 0.1 mM, DBMIB inhibits the pho ments. To identify the steady state, currents were measured in tocurrent completely, whereas upon increasing the concen continuous light and dark conditions without cycling. FIG. 23 tration to 1 mM, the generation of photocurrent is enhanced shows the overlay of light on-off cycle results with that of significantly compared to that without inhibitor. It has also steady state studies at continuous light and continuous dark been observed that a low concentration of 0.01 mM DBMIB conditions. As seen in FIG. 23, over time, the currents from is not sufficient enough to arrest the electron flow at Cyt bef both experiments reached the same steady value. The steady complex. Due to the complete reduction in photocurrent at 0.1 state photocurrent density for Nostoc sp. was 10 mA m. mM DBMIB, it is believed that the Cyt bef is the site in PETC through which the electrons reach the electrode via MWCNT 0185. The major pigment present in all known photosyn generating photocurrent. thetic organisms is chlorophyll a, which forms the reaction centers in both PSII (P680) and PSI (P700), absorbing light (0189 Inhibition of photocurrent by KCN is considerable efficiently at 465 nm and 665 nm. Additionally, cyanobacteria (FIG. 29) with nearly 50% reduction in photocurrent com Such as AV and Nostoc also possess certain accessory pig pared to the wild type. If the major detour of electron towards ments such as phycocyanin, phycoerythrin and carotenoids the electrode is through Cyt bef, the inhibitors downstream of that maximize the range of action spectrum by absorbing a it should not diminish the photocurrent. However, it is not so range of wavelengths other than that absorbed by Chlorophyll in the case of KCN, which indicates that, the total photocur a (Chla). Upon absorbing the characteristic light, these acces rent has not exclusively been arising from the electron leaving sory pigments transfer the absorbed energy to other pigments Cyt befsite. Rather, an alternate pathway could be involved in and finally to the reaction center Chla. FIG. 24A shows the contributing to the photocurrent. The cyclic electron transport action spectrum in the visible region for Nostoc sp. and AV. (CET) around PSI can be one other contributing factor for the containing distinctive peaks corresponding to absorbance for total photocurrent. Experiments with chemicals inhibiting the Chla, phycocyanin, and phycoerythrin. Various insights can CET such as antimycin A will be helpful to analyze contri be achieved by analyzing the generation of photocurrent in bution of cyclic electron transport to the generation of pho Nostoc sp. by illuminating with lights of different character tOCurrent. istic wavelengths, so that the contribution of different pig 0190. The Q cycle catalyzed by Q and Q, sites of Cyt bef ments towards photocurrent generation can be studied. complex represent another possible source for the photocur Experiments have been conducted using lights of 440, 500, rent. The two electrons coming from the oxidation of PQH at 550, 600, 640 and 680 nm wavelengths as shown in FIG.24B. Qo site of Cyt bef complex are not completely transferred to It has been found that Nostoc sp. illuminated using 640 nm PC: rather only one electron is transferred through Cyt f to light (corresponding to phycocyanin) resulted in maximum PC, and the other electron is used to reduce PQ at Q, site photocurrent. through atypical heme X (Zhang et al., 2004; Stroebel et al. 2003). It has been investigated that this hemex near the Q, site 0186 The mechanism of electron transfer from the pho functions as a redox wire allowing ferredoxin or other elec tosynthetic electron transfer chain (PETC) to the MWCNT tron carrier to reduce PQ pool. was also studied with the help of photosystem inhibitors. 0191 The foregoing examples are put forth so as to pro Inhibitors such as DCMU ((3-(3,4-dichlorophenyl)-1,1-dim vide those of ordinary skill in the art with a complete disclo ethylurea), DBMIB (2,5-dibromo-3-methyl-6-isopropylben Sure and description of how to perform the methods and use Zoquinone), KCN (potassium cyanide) and antimycin-A spe the compositions and compounds disclosed and claimed cifically block a particular site of the electron transfer chain herein. Efforts have been made to ensure accuracy with (FIG. 25). DCMU binds at Q site of PSII and inhibit the respect to numbers (e.g., amounts, temperature, etc.), but electron transfer downstream of PSII. DBMIB is an analogue some errors and deviations should be accounted for. Unless of PQ (plastoquinone) and binds at the Qo site of cytochrome indicated otherwise, parts are parts by weight, temperature is bef complex, arresting the electron flow beyond that complex. in C., and pressure is in atmospheres. Standard temperature KCN has been found to replace the copperion of plastocyanin and pressure are defined as 25°C. and 1 atmosphere. (PC), thereby preventing the electron flow from Cyt bef to 0.192 It should be noted that ratios, concentrations, PSI. Antimycin-A blocks the electron transfer from ferre amounts, and other numerical data may be expressed herein doxin to PO, disrupting the cyclic electron transfer around in a range format. It is to be understood that Such a range PSI. All these inhibitors are highly site-specific and have been format is used for convenience and brevity, and thus, should used in the photosynthesis research to decipher the source of be interpreted in a flexible manner to include not only the electron channeling from the photosynthetic machinery. numerical values explicitly recited as the limits of the range, 0187 Experiments were conducted to measure the photo but also to include all the individual numerical values or current produced by the Nostoc sp. after incubating the cells Sub-ranges encompassed within that range as if each numeri with the inhibitors such as DCMU, DBMIB and KCN at cal value and Sub-range is explicitly recited. To illustrate, a varying concentrations, and the results have been Summa concentration range of “about 0.1% to about 5% should be rized in FIG. 26. DCMU was found to inhibit the photocurrent interpreted to include not only the explicitly recited concen by around 80% compared to that of wild type as shown in tration of about 0.1 wt % to about 5 wt %, but also include FIG. 27, indicating that the primary source of photocurrent is individual concentrations (e.g., 1%, 2%. 3%, and 4%) and the the electron from oxidation of water by PSII. The remaining sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within 20% photocurrent could be attributed to either lack of com the indicated range. In an embodiment, the term “about can US 2014/003 8065 A1 Feb. 6, 2014 include traditional rounding according to measurement tech 11. The photosynthetic electrochemical cell of claim 9. niques and the numerical value. In addition, the phrase “about wherein the mediator is selected from the group of redox x to “y” includes “about x to about y”.999 mediators consisting of ferricyanide, a quinone-based com 0193 Many variations and modifications may be made to pound, an osmium complex based compound, a redox chemi the embodiments described in the preceding Examples. All cal compound, and combinations thereof. Such modifications and variations are intended to be included 12. The photosynthetic electrochemical cell of claim 1, herein within the scope of this disclosure and protected by the wherein the anode and cathode comprise conducting materi following claims. als. We claim: 13. The photosynthetic electrochemical cell of claim 1, 1. A photosynthetic electrochemical cell comprising: wherein the anode and cathode conducting materials are an anode composite comprising an anode, a photosynthetic selected from the group of materials consisting of carbon, reaction center (PSRC) including at least one photosyn metal, semiconductor, and combinations thereof, wherein the thetic compound, and a nanostructured material in elec conducting materials are in bulk form nanostructure form, or trochemical communication with the PSRC, wherein the a combination thereof. PSRC is capable of oxidizing water molecules and gen 14. The photosynthetic electrochemical cell of claim 1, erating electrons using a light induced photo-electro wherein the cathode composite further comprises a matrix of chemical reaction and wherein at least a portion of elec nanostructured material coupling the at least one enzyme or trons generated by the PSRC are transferred to the anode metallic catalyst capable of reducing the reductant to the via direct electron transfer; and cathode. a cathode composite comprising a cathode and at least one 15. The photosynthetic electrochemical cell of claim 1, enzyme or metallic catalyst capable of reducing a reduc wherein the PSRC is coupled to the nanostructured material tant. by a linking agent. 2. The photosynthetic electrochemical cell of claim 1, 16. The photosynthetic electrochemical cell of claim 1, wherein the PSRC includes at lest one photosynthetic protein wherein the linking agentis selected from the group of linking selected from the group consisting of PSII, PSI, cytochrome agents consisting of 1-pyrenebutanoic acid Succinimidyl bef (Cyt-bf), plastocyanin, and combinations thereof. ester (PBSE), a protein homo-bifunctional cross-linking 3. The photosynthetic electrochemical cell of claim 1, agent, a hetero-bifunctional cross-linking agent, and combi wherein the PSRC comprises PSII and further comprises at nations thereof. least one photosynthetic compound selected from the group 17. A photosynthetic electrochemical cell comprising: consisting of PSI, plastoquinone, cyt bef, plastocyanin, phy an anode composite comprising an anode in electrochemi cocyanin, phycoerythrin, a carotenoid compound, and com cal communication with a thylakoid membrane, wherein binations thereof. the thylakoid membrane is capable of oxidizing water 4. The photosynthetic electrochemical cell of claim 1, wherein the PSRC comprises at least two photosynthetic molecules and generating electrons using light induced compounds selected from the group consisting of PSII, PSI, photo-electrochemical reactions, wherein the anode plastoquinone, cyt bef, plastocyanin, phycocyanin, phyco composite is configured Such that electrons generated by erythrin, a carotenoid compound, and combinations thereof. the thylakoid membrane are conducted to the anode via 5. The photosynthetic electrochemical cell of claim 1, direct electron transfer; and wherein the nanostructured material comprises matrix of a cathode composite comprising a cathode and at least one nanostructured material and wherein the matrix of nanostruc enzyme or metallic catalyst capable of reducing O. ture material couples the PSRC to the anode. 18. The photosynthetic electrochemical cell of claim 17, 6. The photosynthetic electrochemical cell of claim 5, wherein the thylakoid membrane is coupled to the anode by a wherein the matrix of nanostructured materials is selected matrix of nanostructured material. from the group of nanostructured materials consisting of 19. The photosynthetic electrochemical cell of claim 18, carbon nanostructured materials, metallic nanoparticles, wherein the matrix of nanostructured materials is selected semiconductor nanoparticles, quantum dots and combina from the group of nanostructured materials consisting of tions of these materials. carbon nanotubes, multi-walled carbon nanotubes, 7. The photosynthetic electrochemical cell of claim 6, fullerenes, carbon nanoparticles, graphenes, two-dimen wherein the carbon nanostructured materials are selected sional carbon nanosheets, graphite platelets, other carbon from the group of carbon nanostructures consisting of carbon nanostructured materials, metallic nanoparticles, semicon nanotubes, multi-walled carbon nanotubes, fullerenes, car ductor nanoparticles, quantum dots, and combinations of bon nanoparticles, graphenes, two-dimensional carbon these materials. nanosheets, graphite platelets, and combinations of these 20. The photosynthetic electrochemical cell of claim 17, materials. wherein the thylakoid membrane is part of an intact thylakoid 8. The photosynthetic electrochemical cell of claim 5, organelle. wherein the matrix of nanostructured material comprises 21. The photosynthetic electrochemical cell of claim 17, multi-walled carbon nanotubes. wherein the thylakoid membrane is coupled to the matrix of 9. The photosynthetic electrochemical cell of claim 1, nanostructured material by a linking agent. wherein the reductant is O and the at least one enzyme 22. The photosynthetic electrochemical cell of claim 21, capable of reducing O is selected from the group consisting wherein the linking agentis selected from the group of linking of laccase, bilirubin oxidase, ascorbate oxidase, tyrosinase, agents consisting of 1-pyrenebutanoic acid Succinimidyl catechol oxidase, and combinations thereof. ester (PBSE), a protein homo-bifunctional cross-linking 10. The photosynthetic electrochemical cell of claim 1, agent, a hetero-bifunctional cross-linking agent, and combi further comprising a redox mediator. nations thereof. US 2014/003 8065 A1 Feb. 6, 2014
23. The photosynthetic electrochemical cell of claim 17, includes one or more of the following photosynthetic com wherein they thylakoid membrane includes at least two of the pounds: phycocyanin, phycoerythrin, and a carotenoid com following photosynthetic compounds: PSII, plastoquinone, pound. cyt bef, plastocyanin, and PSI. 35. The photosynthetic electrochemical cell of claim 28, 24. The photosynthetic electrochemical cell of claim 17, wherein the photosynthetic organism or part thereof is wherein the at least one enzyme capable of reducing O is coupled to the matrix of nanostructured material by a linking selected from the group consisting of laccase, bilirubin oxi agent selected from the group consisting of 1-pyrenebu dase, ascorbate oxidase, tyrosinase, catechol oxidase, and tanoic acid succinimidyl ester (PBSE), a protein homo-bi combinations thereof. functional cross-linking agent, a hetero-bifunctional cross 25. The photosynthetic electrochemical cell of claim 17, linking agents, and a combination thereof. wherein the cathode composite further comprises a matrix of 36. The photosynthetic electrochemical cell of claim 28, nanostructured material coupling the at least one enzyme or wherein the at least one enzyme capable of reducing O is metallic catalyst capable of reducing O to the cathode. selected from the group consisting of laccase, bilirubin oxi 26. The photosynthetic electrochemical cell of claim 17, dase, ascorbate oxidase, tyrosinase, catechol oxidase, and a further comprising a redox mediator. combination thereof. 27. The photosynthetic electrochemical cell of claim 26, 37. The photosynthetic electrochemical cell of claim 28, wherein the mediatoris selected from the group consisting of further comprising a redox mediator. ferricyanide, a quinone based compound, an osmium com 38. The photosynthetic electrochemical cell of claim 37, wherein the mediatoris selected from the group consisting of plex based compound, aredox chemical compound, and com ferricyanide, a quinone based compound, an osmium com binations thereof. plex based compound, a redox chemical compound, and a 28. A photosynthetic electrochemical cell comprising: combination thereof. an anode composite comprising an anode in electrochemi 39. A method of generating an electrical current compris cal communication with a photosynthetic organism or a ing: part of a photosynthetic organism, wherein the photo synthetic organism or part thereof is capable of oxidiz providing an electrochemical cell comprising: ing water molecules and generating electrons using light an anode composite having photosynthetic reaction cen induced photo-electrochemical reactions and wherein ters (PSRC), wherein the PSRCs include at least one the anode composite is configured such that at least photosynthetic compound and the PSRCs are in elec Some electrons generated by the photosynthetic organ trical communication with an anode via a nanostruc ism or part thereofare conducted to the anode via direct tured material, and electron transfer, and a cathode composite capable of reducing O, and exposing the electrochemical cell to light in the presence of a cathode composite comprising a cathode and at least one water, wherein the PSRC uses light energy to oxidize a enzyme or metallic catalyst capable of reducing O. water molecule and generate electrons, which are trans 29. The photosynthetic electrochemical cell of claim 28, ferred to the anode via the nanostructured material, and wherein the photosynthetic organism comprises one or more wherein electrons generated at the anode reduce O at a photosynthetic organisms selected from the group of photo cathode, thereby inducing a potential difference synthetic organisms consisting of cyanobacteria, green Sul between the anode and the cathode and generating an fur bacteria, algae, Spirulina, chlorella, and combinations electrical current. thereof. 40. The method of claim 39, wherein the photosynthetic 30. The photosynthetic electrochemical cell of claim 28, reaction centers comprise at least one photosynthetic protein wherein the photosynthetic organism is selected from the selected from the group consisting of PSII, PSI, cytochrome group consisting of Nostoc sp., Anabaena variabilis, Syn bef (Cytbf), plastocyanin, and combinations thereof. echococcus sp., Spirulina sp., Rhobacter sp., Rhodobium sp., 41. The method of claim 39, wherein the photosynthetic Chlorobium sp., and combinations thereof. PSRC comprises PSII and at least one photosynthetic com 31. The photosynthetic electrochemical cell of claim 28, pound selected from the group consisting of PSI, plasto wherein the photosynthetic organism or part thereof is quinone, cyt bef, plastocyanin, phycocyanin, phycoerythrin, a coupled to the anode by a matrix of nanostructured material. carotenoid compound, and combinations thereof. 32. The photosynthetic electrochemical cell of claim 31, 42. The method of claim 39, wherein the nanostructured wherein the matrix of nanostructured materials is selected material comprises a matrix of nanostructured material and from the group of nanostructured materials consisting of wherein the matrix of nanostructure material couples the carbon nanotubes, multi-walled carbon nanotubes, PSRC to the anode. fullerenes, carbon nanoparticles, graphenes, two-dimen 43. The method of claim 42, wherein the matrix of nano sional carbon nanosheets, graphite platelets, other carbon structured materials is selected from the group of nanostruc nanostructured materials, metallic nanoparticles, semicon tured materials consisting of carbon nanotubes, multi-walled ductor nanoparticles, quantum dots, and combinations of carbon nanotubes, fullerenes, carbon nanoparticles, these materials. graphenes, two-dimensional carbon nanosheets, graphite 33. The photosynthetic electrochemical cell of claim 23, platelets, other carbon nanostructured materials, metallic wherein the photosynthetic organism or part thereof includes nanoparticles, semiconductor nanoparticles, quantum dots, at least two the following photosynthetic compounds: PSII, and combinations of these materials. plastoquinone, cyt bef, plastocyanin, and PSI. 44. The method of claim 39, wherein the cathode compos 34. The photosynthetic electrochemical cell of claim 28, ite comprises at least one enzyme capable of reducing O. wherein the photosynthetic organism or part thereof further wherein Such enzyme is selected from the group consisting US 2014/003 8065 A1 Feb. 6, 2014 18 of laccase, bilirubin oxidase, ascorbate oxidase, tyrosinase, catechol oxidase, and combinations thereof. 45. The method of claim 39, wherein the electrochemical cell further comprises a redox mediator. 46. A method of generating an electrical current compris ing converting light energy to electrical energy using a pho tosynthetic electrochemical cell comprising: an anode composite comprising an anode, at least one photosynthetic reaction center (PSRC) including at least one photosynthetic compound, and a nanostructured material in electrochemical communication with the at least one PSRC, wherein the PSRC is capable of oxidiz ing water molecules and generating electrons using a light induced photo-electrochemical reaction and wherein electrons generated by the PSRC are transferred to the anode via direct electron transfer; and a cathode composite comprising a cathode and at least one enzyme or metallic catalyst capable of reducing O. k k k k k