U.S. patent application number 13/957494 was filed with the patent office on 2014-02-06 for photosynthetic electrochemical cells.
The applicant listed for this patent is University of Georgia Research Foundation, Inc.. Invention is credited to Ramaraja P. Ramasamy.
Application Number | 20140038065 13/957494 |
Document ID | / |
Family ID | 50025805 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038065 |
Kind Code |
A1 |
Ramasamy; Ramaraja P. |
February 6, 2014 |
PHOTOSYNTHETIC ELECTROCHEMICAL CELLS
Abstract
The present disclosure provides photosynthetic electrochemical
cells including photosynthetic compounds and methods of generating
an electrical current using the photosynthetic electrochemical
cells.
Inventors: |
Ramasamy; Ramaraja P.;
(Watkinsville, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Georgia Research Foundation, Inc. |
Athens |
GA |
US |
|
|
Family ID: |
50025805 |
Appl. No.: |
13/957494 |
Filed: |
August 2, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61679118 |
Aug 3, 2012 |
|
|
|
Current U.S.
Class: |
429/401 ;
429/532 |
Current CPC
Class: |
H01L 51/0093 20130101;
Y02E 60/50 20130101; H01G 9/2059 20130101; H01M 14/005 20130101;
Y02E 10/542 20130101; H01L 51/0048 20130101; B82Y 10/00 20130101;
H01M 8/16 20130101 |
Class at
Publication: |
429/401 ;
429/532 |
International
Class: |
H01M 14/00 20060101
H01M014/00 |
Claims
1. A photosynthetic electrochemical cell comprising: an anode
composite comprising an anode, a photosynthetic reaction center
(PSRC) including at least one photosynthetic compound, and a
nanostructured material in electrochemical communication with the
PSRC, wherein the PSRC is capable of oxidizing water molecules and
generating electrons using a light induced photo-electrochemical
reaction and wherein at least a portion of 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 a reductant.
2. The photosynthetic electrochemical cell of claim 1, wherein the
PSRC includes at lest one photosynthetic protein selected from the
group consisting of: PSII, PSI, cytochrome b.sub.6f (Cyt-b.sub.6f),
plastocyanin, and combinations thereof.
3. The photosynthetic electrochemical cell of claim 1, wherein the
PSRC comprises PSII and further comprises at least one
photosynthetic compound selected from the group consisting of: PSI,
plastoquinone, cyt b.sub.6f, plastocyanin, phycocyanin,
phycoerythrin, a carotenoid compound, and combinations thereof.
4. The photosynthetic electrochemical cell of claim 1, wherein the
PSRC comprises at least two photosynthetic compounds selected from
the group consisting of: PSII, PSI, plastoquinone, cyt b.sub.6f,
plastocyanin, phycocyanin, phycoerythrin, a carotenoid compound,
and combinations thereof.
5. The photosynthetic electrochemical cell of claim 1, wherein the
nanostructured material comprises matrix of nanostructured material
and wherein the matrix of nanostructure material couples the PSRC
to the anode.
6. The photosynthetic electrochemical cell of claim 5, wherein the
matrix of nanostructured materials is selected from the group of
nanostructured materials consisting of: carbon nanostructured
materials, metallic nanoparticles, semiconductor nanoparticles,
quantum dots and combinations of these materials.
7. The photosynthetic electrochemical cell of claim 6, wherein the
carbon nanostructured materials are selected from the group of
carbon nanostructures consisting of: carbon nanotubes, multi-walled
carbon nanotubes, fullerenes, carbon nanoparticles, graphenes,
two-dimensional carbon nanosheets, graphite platelets, and
combinations of these materials.
8. The photosynthetic electrochemical cell of claim 5, wherein the
matrix of nanostructured material comprises multi-walled carbon
nanotubes.
9. The photosynthetic electrochemical cell of claim 1, wherein the
reductant is O.sub.2 and the at least one enzyme capable of
reducing O.sub.2 is selected from the group consisting of: laccase,
bilirubin oxidase, ascorbate oxidase, tyrosinase, catechol oxidase,
and combinations thereof.
10. The photosynthetic electrochemical cell of claim 1, further
comprising a redox mediator.
11. The photosynthetic electrochemical cell of claim 9, wherein the
mediator is selected from the group of redox mediators consisting
of: ferricyanide, a quinone-based compound, an osmium complex based
compound, a redox chemical compound, and combinations thereof.
12. The photosynthetic electrochemical cell of claim 1, wherein the
anode and cathode comprise conducting materials.
13. The photosynthetic electrochemical cell of claim 1, wherein the
anode and cathode conducting materials are selected from the group
of materials consisting of: carbon, metal, semiconductor, and
combinations thereof, wherein the conducting materials are in bulk
form nanostructure form, or a combination thereof.
14. The photosynthetic electrochemical cell of claim 1, wherein the
cathode composite further comprises a matrix of nanostructured
material coupling the at least one enzyme or metallic catalyst
capable of reducing the reductant to the cathode.
15. The photosynthetic electrochemical cell of claim 1, wherein the
PSRC is coupled to the nanostructured material by a linking
agent.
16. The photosynthetic electrochemical cell of claim 1, wherein the
linking agent is selected from the group of linking agents
consisting of: 1-pyrenebutanoic acid succinimidyl ester (PBSE), a
protein homo-bifunctional cross-linking agent, a
hetero-bifunctional cross-linking agent, and combinations
thereof.
17. A photosynthetic electrochemical cell comprising: an anode
composite comprising an anode in electrochemical communication with
a thylakoid membrane, wherein the thylakoid membrane is capable of
oxidizing water molecules and generating electrons using light
induced photo-electrochemical reactions, wherein the anode
composite is configured such that electrons generated by the
thylakoid membrane are conducted 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.sub.2.
18. The photosynthetic electrochemical cell of claim 17, wherein
the thylakoid membrane is coupled to the anode by a matrix of
nanostructured material.
19. The photosynthetic electrochemical cell of claim 18, wherein
the matrix of nanostructured materials is selected from the group
of nanostructured materials consisting of: carbon nanotubes,
multi-walled carbon nanotubes, fullerenes, carbon nanoparticles,
graphenes, two-dimensional carbon nanosheets, graphite platelets,
other carbon nanostructured materials, metallic nanoparticles,
semiconductor nanoparticles, quantum dots, and combinations of
these materials.
20. The photosynthetic electrochemical cell of claim 17, wherein
the thylakoid membrane is part of an intact thylakoid
organelle.
21. The photosynthetic electrochemical cell of claim 17, wherein
the thylakoid membrane is coupled to the matrix of nanostructured
material by a linking agent.
22. The photosynthetic electrochemical cell of claim 21, wherein
the linking agent is selected from the group of linking agents
consisting of: 1-pyrenebutanoic acid succinimidyl ester (PBSE), a
protein homo-bifunctional cross-linking agent, a
hetero-bifunctional cross-linking agent, and combinations
thereof.
23. The photosynthetic electrochemical cell of claim 17, wherein
they thylakoid membrane includes at least two of the following
photosynthetic compounds: PSII, plastoquinone, cyt b.sub.6f,
plastocyanin, and PSI.
24. The photosynthetic electrochemical cell of claim 17, wherein
the at least one enzyme capable of reducing O.sub.2 is selected
from the group consisting of: laccase, bilirubin oxidase, ascorbate
oxidase, tyrosinase, catechol oxidase, and combinations
thereof.
25. The photosynthetic electrochemical cell of claim 17, wherein
the cathode composite further comprises a matrix of nanostructured
material coupling the at least one enzyme or metallic catalyst
capable of reducing O.sub.2 to the cathode.
26. The photosynthetic electrochemical cell of claim 17, further
comprising a redox mediator.
27. The photosynthetic electrochemical cell of claim 26, wherein
the mediator is selected from the group consisting of:
ferricyanide, a quinone based compound, an osmium complex based
compound, a redox chemical compound, and combinations thereof.
28. A photosynthetic electrochemical cell comprising: an anode
composite comprising an anode in electrochemical communication with
a photosynthetic organism or a part of a photosynthetic organism,
wherein the photosynthetic organism or part thereof is capable of
oxidizing water molecules and generating electrons using light
induced photo-electrochemical reactions and wherein the anode
composite is configured such that at least some electrons generated
by the photosynthetic organism or part thereof are conducted 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.sub.2.
29. The photosynthetic electrochemical cell of claim 28, wherein
the photosynthetic organism comprises one or more photosynthetic
organisms selected from the group of photosynthetic organisms
consisting of: cyanobacteria, green sulfur bacteria, algae,
spirulina, chlorella, and combinations thereof.
30. The photosynthetic electrochemical cell of claim 28, wherein
the photosynthetic organism is selected from the group consisting
of: Nostoc sp., Anabaena variabilis, Synechococcus sp., Spirulina
sp., Rhobacter sp., Rhodobium sp., Chlorobium sp., and combinations
thereof.
31. The photosynthetic electrochemical cell of claim 28, wherein
the photosynthetic organism or part thereof is coupled to the anode
by a matrix of nanostructured material.
32. The photosynthetic electrochemical cell of claim 31, wherein
the matrix of nanostructured materials is selected from the group
of nanostructured materials consisting of: carbon nanotubes,
multi-walled carbon nanotubes, fullerenes, carbon nanoparticles,
graphenes, two-dimensional carbon nanosheets, graphite platelets,
other carbon nanostructured materials, metallic nanoparticles,
semiconductor nanoparticles, quantum dots, and combinations of
these materials.
33. The photosynthetic electrochemical cell of claim 23, wherein
the photosynthetic organism or part thereof includes at least two
the following photosynthetic compounds: PSII, plastoquinone, cyt
b.sub.6f, plastocyanin, and PSI.
34. The photosynthetic electrochemical cell of claim 28, wherein
the photosynthetic organism or part thereof further includes one or
more of the following photosynthetic compounds: phycocyanin,
phycoerythrin, and a carotenoid compound.
35. The photosynthetic electrochemical cell of claim 28, wherein
the photosynthetic organism or part thereof is coupled to the
matrix of nanostructured material by a linking agent selected from
the group consisting of: 1-pyrenebutanoic acid succinimidyl ester
(PBSE), a protein homo-bifunctional cross-linking agent, a
hetero-bifunctional cross-linking agents, and a combination
thereof.
36. The photosynthetic electrochemical cell of claim 28, wherein
the at least one enzyme capable of reducing O.sub.2 is selected
from the group consisting of: laccase, bilirubin oxidase, ascorbate
oxidase, tyrosinase, catechol oxidase, and a combination
thereof.
37. The photosynthetic electrochemical cell of claim 28, further
comprising a redox mediator.
38. The photosynthetic electrochemical cell of claim 37, wherein
the mediator is selected from the group consisting of:
ferricyanide, a quinone based compound, an osmium complex based
compound, a redox chemical compound, and a combination thereof.
39. A method of generating an electrical current comprising:
providing an electrochemical cell comprising: an anode composite
having photosynthetic reaction centers (PSRC), wherein the PSRCs
include at least one photosynthetic compound and the PSRCs are in
electrical communication with an anode via a nanostructured
material, and a cathode composite capable of reducing O.sub.2; and
exposing the electrochemical cell to light in the presence of
water, wherein the PSRC uses light energy to oxidize a water
molecule and generate electrons, which are transferred to the anode
via the nanostructured material, and wherein electrons generated at
the anode reduce O.sub.2 at a cathode, thereby inducing a potential
difference between the anode and the cathode and generating an
electrical current.
40. The method of claim 39, wherein the photosynthetic reaction
centers comprise at least one photosynthetic protein selected from
the group consisting of: PSII, PSI, cytochrome b.sub.6f
(Cytb.sub.6f), plastocyanin, and combinations thereof.
41. The method of claim 39, wherein the photosynthetic PSRC
comprises PSII and at least one photosynthetic compound selected
from the group consisting of: PSI, plastoquinone, cyt b.sub.6f,
plastocyanin, phycocyanin, phycoerythrin, a carotenoid compound,
and combinations thereof.
42. The method of claim 39, wherein the nanostructured material
comprises a matrix of nanostructured material and wherein the
matrix of nanostructure material couples the PSRC to the anode.
43. The method of claim 42, wherein the matrix of nanostructured
materials is selected from the group of nanostructured materials
consisting of: carbon nanotubes, multi-walled carbon nanotubes,
fullerenes, carbon nanoparticles, graphenes, two-dimensional carbon
nanosheets, graphite platelets, other carbon nanostructured
materials, metallic nanoparticles, semiconductor nanoparticles,
quantum dots, and combinations of these materials.
44. The method of claim 39, wherein the cathode composite comprises
at least one enzyme capable of reducing O.sub.2, wherein such
enzyme is selected from the group consisting 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 comprising
converting light energy to electrical energy using a photosynthetic
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 oxidizing 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.sub.2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to copending U.S.
provisional application entitled, "Photosynthetic electrochemical
cells," having Ser. No. 61/679,118, filed Aug. 3, 2012, which is
entirely incorporated herein by reference.
BACKGROUND
[0002] Plant photosynthesis provides an unmatched quantum
efficiency of nearly 100%. In recent years, significant interest
has evolved in mimicking and/or harnessing the photosynthetic
process for energy conversion and hydrogen generation applications.
Multiple approaches to artificial photosynthesis exist, including
light energy harvesting using natural pigments from plants and
microorganisms and using whole cell microorganisms. Scientists have
explored photosynthetic organelles such as thylakoids, chlorophyll
molecules, photosystems, and oxygen evolving complexes for
photo-electrochemical activity. However, the challenge of
establishing electrical communication between photosynthetic
reaction centers and the electrode has proven extremely difficult.
Thus, to date, a photosynthetic electrochemical cell that allows
direct electron transfer between the photosynthetic centers and an
electrode has remained elusive.
SUMMARY
[0003] Briefly described, embodiments of the present disclosure
provide for photosynthetic electrochemical cells and methods of
using the photosynthetic electrochemical cells to produce an
electrical current.
[0004] In embodiments, photosynthetic electrochemical cells of the
present disclosure include an anode composite having an anode, a
photosynthetic reaction center (PSRC) including at least one
photosynthetic compound, and a nanostructured material in
electrochemical communication with the PSRC, and a cathode
composite having a cathode and at least one enzyme or metallic
catalyst capable of reducing O.sub.2. The PSRC is capable of
oxidizing water molecules and generating electrons using a light
induced photo-electrochemical reaction, and the electrons generated
by the PSRC are transferred to the anode via direct electron
transfer. In embodiments, the photosynthetic compounds can include,
but are not limited to, PSII, PSI, plastoquinone, cyt b.sub.6f,
plastocyanin, phycocyanin, phycoerythrin, carotenoids, and
combinations of these compounds. In embodiments, the nanostructured
material is a matrix of nanostructured materials which can be made
from materials such as, but not limited to, carbon nanotubes,
multi-walled carbon nanotubes, fullerenes, carbon nanoparticles,
graphenes, carbon nanosheets, two-dimensional carbon platelets,
other carbon nanostructured materials, metallic nanoparticles,
semiconductor nanoparticles, quantum dots, and combinations of
these materials.
[0005] The present disclosure also includes embodiments of
photosynthetic electrochemical cells including an anode composite
having an anode in electrochemical communication with a thylakoid
membrane, and a cathode composite having a cathode and at least one
enzyme or metallic catalyst capable of reducing O.sub.2. The
thylakoid membrane in such embodiments is capable of oxidizing
water molecules and generating electrons using light induced
photo-electrochemical reactions, and the anode composite is
configured such that electrons generated by the thylakoid membrane
are conducted to the anode via direct electron transfer. In some
embodiments, the thylakoid membrane is coupled to the anode by a
matrix of nanostructured material, such as described above.
[0006] Additional embodiments of photosynthetic electrochemical
cells of the present disclosure include an anode composite having
an anode in electrochemical communication with a photosynthetic
organism or a part of a photosynthetic organism, and a cathode
composite having a cathode and at least one enzyme or metallic
catalyst capable of reducing O.sub.2. In such embodiments, the
photosynthetic organism or part thereof is capable of oxidizing
water molecules and generating electrons using light induced
photo-electrochemical reactions, and the anode composite is
configured such that the electrons generated by the photosynthetic
organism or part thereof are conducted to the anode via direct
electron transfer. In some embodiments, the photosynthetic organism
or part thereof is coupled to the anode by a matrix of
nanostructured material, as described above. In embodiments, the
photosynthetic organism can include, but is not limited to,
cyanobacteria, green sulfur bacteria, algae, spirulina, chlorella,
and combinations of such organisms.
[0007] The present disclosure also includes methods of generating
an electrical current with a photosynthetic electrochemical cell.
In embodiments, methods of generating an electrical current include
providing an electrochemical cell including an anode composite
having photosynthetic reaction centers (PSRC) that include at least
one photosynthetic compound and are in electrical communication
with an anode via a nanostructured material and a cathode composite
capable of reducing O.sub.2; and exposing the electrochemical cell
to light in the presence of water. In such methods, the PSRCs of
the anode composite use light energy to oxidize water molecules and
generate electrons, which are transferred to the anode via the
nanostructured material, and then reduce O.sub.2 at a cathode,
thereby inducing a potential difference between the anode and the
cathode and generating an electrical current.
[0008] Embodiments of methods of generating an electrical current
of the present disclosure also include converting light energy to
electrical energy using a photosynthetic electrochemical cell of
the present disclosure.
[0009] Other systems, methods, features, and advantages of the
present disclosure will be or become apparent to one with skill in
the art upon examination of the following drawings and detailed
description. It is intended that all such additional systems,
methods, features, and advantages be included within this
description, and be within the scope of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The disclosure can be better understood with reference to
the following drawings, which are discussed in the description and
examples below. The components in the drawings are not necessarily
to scale, emphasis instead being placed upon clearly illustrating
the principles of the present disclosure.
[0011] FIG. 1 illustrates a schematic representation of a thylakoid
membrane tethered to MWNT modified electrode using PBSE linkers and
the likely electron transport pathways [(a), (b) and (c)] between
thylakoid membrane proteins and the electrode. OEC, PQ, Cyt, PC,
FD, and ATP Syn represent oxygen evolving complex, plastoquinone,
cytochrome, plastocyanin, ferredoxin and ATP synthase,
respectively. PSI and PSII represent the photosynthetic reaction
centers I and II, respectively. Other components of the thylakoid
membrane are not shown.
[0012] FIG. 2 is a group of digital AFM images of gold electrodes
modified with (a) MWNT without thylakoid (b) thylakoid-MWNT
composite and (c) thylakoid without MWNT. The topography,
amplitude, and phase images correspond to left, middle and right
columns respectively. Thylakoids are marked by the arrows in the
images.
[0013] FIGS. 3A-3B are a group of SEM images of bare electrode
modified with (3A) thylakoids, (3B) MWNT and (3C) thylakoids-MWNT
composite. Thylakoids are shown by the arrows.
[0014] FIGS. 4A-4B are graphs of cyclic voltammograms (CV) of a
thylakoid-MWNT composite modified electrode. FIG. 4A illustrates
the CV in the presence and absence of 1.5 mM mediator; inset graph
shows capacitance subtracted voltammogram. FIG. 4B illustrates the
CV under light and dark conditions with 1.5 mM mediator; inset
graph shows the background subtracted voltammograms. 1.sub.peak,
2.sub.peak and 3.sub.peak represent the redox reactions of cyt b6f,
ferricyanide mediator, and plastocyanin, respectively.
[0015] FIG. 5A is a graph of pen circuit potentials of unmodified
(control) and thylakoid modified MWNT electrodes in the presence of
1.5 mM [Fe(CN).sub.6].sup.3-/4-. FIG. 5B illustrates a graph of
photo-current responses of unmodified (control) and thylakoid
modified MWNT electrodes at a fixed potential of 0.2 V in the
presence of 1.5 mM [Fe(CN).sub.6].sup.3-/4-.
[0016] FIG. 5C illustrates Nyquist plots (Z' vs. -Z'') for
thylakoid-MWNT composites under light and dark conditions. Inset in
FIG. 5C shows the equivalent circuit model used to fit the Nyquist
data. In FIGS. 5A and 5B, .uparw. and .dwnarw. represent light on
and light off conditions, respectively.
[0017] FIG. 6 is a graph illustrating a comparison of the
photo-current responses of unexposed and DCMU exposed
thylakoid-MWNT composites. .uparw. and .dwnarw. represent light on
and light off conditions, respectively.
[0018] FIG. 7A illustrates a schematic representation of an
embodiments of a photo-electrochemical cell of the present
disclosure containing thylakoid-MWNT based photo-anode and
laccase-MWNT based biocathode. FIG. 7B is a graph of the steady
state polarization and power density curves of the
photo-electrochemical cell. A digital image of the simple
photo-electrochemical cell setup is shown in the inset.
[0019] FIG. 8 illustrates a UV-Vis spectrum of suspended thylakoid
membranes used to calculate chlorophyll concentration.
[0020] FIGS. 9A-D are graphs illustrating photocurrent analysis of
thylakoid-MWNT composites for the optimization of (9A) thylakoid
immobilization time, (9B) chlorophyll loading, (9C) mediator
concentration and (9D) applied potential. .uparw. represents light
condition and .dwnarw. represents dark condition. An applied
potential of 0.2 V, 1 hr thylakoid immobilization time, and 1.5 mM
mediator concentration were the most favorable conditions for this
technique.
[0021] FIG. 10 illustrates cyclic voltammograms of thylakoid-MWNT
composites in the presence and absence of 10 mM KCN as plastocyanic
inhibitor.
[0022] FIG. 11 is a graph illustrating photocurrent responses of
1.5 mM 2,6-dichloro-p-benzoquinone and 50 mM 1,4-benzoquinone
mediators on MWNT electrodes in the absence of thylakoids. The
photocurrent response of ferricyanide mediator is also shown for
comparison.
[0023] FIGS. 12A-12D illustrate the absorption spectrum of
thylakoid membranes with exposure to ferricyanide mediator (FIG.
12A), KCN (FIG. 12B), paraquat/diquat (FIG. 12C), and DCMU
herbicide (FIG. 12D).
[0024] FIG. 13 is a graph illustrating the effect of light
intensity on thylakoid-MWNT composites. High light intensity was
turned on at 60 s, medium at 540 s and low light at 920s.
[0025] FIGS. 14A-14B illustrate photocurrent analysis of
thylakoid-MWNT modified gold electrode under: constant light (FIG.
14A) and constant dark (FIG. B) conditions, as compared to
light/dark cycle.
[0026] FIGS. 15A-15B illustrate a comparison of immobilized
thylakoids (1.times.) with various quantities (20.times.,
100.times., 200.times.) of suspended thylakoids in solution. FIG.
15A is a graph of photocurrent response (.uparw. represents light
condition and .dwnarw. represents dark condition), FIG. 15B
illustrates cyclic voltammograms under light. Note: 1.times.
corresponds to 0.014 mg.sub.chl.
[0027] FIG. 16A illustrates cyclic voltammograms of thylakoid-MWNT
composites with and without exposure to DCMU herbicide, and FIG.
16B illustrates background subtracted cyclic voltammograms showing
the retention of redox peaks of cyt-b.sub.6f (0 V) and plastocyanin
(0.2 V).
[0028] FIG. 17 is a graph illustrating a comparison of the
photo-current responses of unexposed and paraquat exposed
thylakoid-MWNT composites. .uparw. and .dwnarw. represents light on
and light off conditions, respectively.
[0029] FIG. 18 is a group of scanning Electron Microscopic images:
(a) carbon paper, (b) carbon paper with MWCNT (CP-MWCNT), (c)
Anabaena variabilis immobilized on CP-MWCNT and (d) Nostoc sp.
immobilized on CP-MWCNT.
[0030] FIG. 19A is a graph illustrating measurement of open circuit
potential comparing bare and PS bacteria-modified CP-MWCNT
electrodes, and the graph in FIG. 19B illustrates photocurrent
density of bare and PS bacteria modified CP-MWCNT.
[0031] FIG. 20 is a graph illustrating dependency of photocurrent
generation on Nostoc sp. loading on the CP-MWCNT electrode.
[0032] FIG. 21 is a graph illustrating dependency of photocurrent
generation on AV loading on the CP-MWCNT electrode.
[0033] FIG. 22 is a graph illustrating photocurrent generation in
Nostoc sp. illuminated with white light of varying intensities.
[0034] FIG. 23 is a graph illustrating the stability of
photocurrent generation in Nostoc sp. under continuous light and
dark and alternate light/dark phases.
[0035] FIGS. 24A and 24B are graphs, with FIG. 24A illustrating
absorption spectrum of Nostoc and A. variablis in the visible light
spectrum, while FIG. 24B illustrates the measurement of
photocurrent of Nostoc sp. at different characteristic
wavelengths.
[0036] FIG. 25 illustrates the photosynthetic electron transport
chain and blocking sites of various inhibitors.
[0037] FIG. 26A is a graph illustrating the effect of various
inhibitors of photosynthetic electron transport chain on
photocurrent generation in Nostoc sp, and FIG. 26B is a bar graph
showing the percentage inhibition of each inhibitor (DCMU, DBMIB,
and KCM)
[0038] FIG. 27A illustrates the effect of varying concentration of
DCMU on photocurrent generation in Nostoc sp. FIG. 27B is a bar
graph illustrating the decrease in percentage photocurrent as a
function of concentration of DCMU.
[0039] FIG. 28 illustrates the effect of varying concentration of
DBMIB on photocurrent generation in Nostoc sp.
[0040] FIG. 29A illustrates the effect of varying concentration of
KCN on photocurrent generation in Nostoc sp. FIG. 29B is a bar
graph illustrating the decrease in percentage photocurrent as a
function of concentration of KCN.
DESCRIPTION
[0041] The details of some embodiments of the present disclosure
are set forth in the description below. Other features, objects,
and advantages of the present disclosure will be apparent to one of
skill in the art upon examination of the following description,
drawings, examples and claims. It is intended that all such
additional systems, methods, features, and advantages be included
within this description, be within the scope of the present
disclosure, and be protected by the accompanying claims
[0042] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, and as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
disclosure will be limited only by the appended claims.
[0043] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0045] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0046] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0047] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of molecular biology, microbiology,
organic chemistry, biochemistry, genetics, botany, electrochemistry
and the like, which are within the skill of the art. Such
techniques are explained fully in the literature.
[0048] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
[0049] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise. In this disclosure,
"comprises," "comprising," "containing" and "having" and the like
can have the meaning ascribed to them in U.S. Patent law and can
mean "includes," "including," and the like; "consisting essentially
of" or "consists essentially" or the like, when applied to methods
and compositions encompassed by the present disclosure refers to
compositions like those disclosed herein, but which may contain
additional structural groups, composition components or method
steps. Such additional structural groups, composition components or
method steps, etc., however, do not materially affect the basic and
novel characteristic(s) of the compositions or methods, compared to
those of the corresponding compositions or methods disclosed
herein. "Consisting essentially of" or "consists essentially" or
the like, when applied to methods and compositions encompassed by
the present disclosure have the meaning ascribed in U.S. Patent law
and the term is open-ended, allowing for the presence of more than
that which is recited so long as basic or novel characteristics of
that which is recited is not changed by the presence of more than
that which is recited, but excludes prior art embodiments.
[0050] Prior to describing the various embodiments, the following
definitions are provided and should be used unless otherwise
indicated.
DEFINITIONS
[0051] In describing and claiming the disclosed subject matter, the
following terminology will be used in accordance with the
definitions set forth below.
[0052] As used herein, the term "photosynthetic compound" includes
any compound involved in the photosynthetic process, e.g., the
process of harnessing light energy to induce a photochemical
reaction to oxidize water molecules and generate electrons.
"Photosynthetic compounds" includes "photosynthetic proteins" and
protein complexes, such as, but not limited to, PSI, PSII, cyt
b.sub.6f, plastocyanin, phycocyanin, and phycoerythrin as well as
other non-protein, photosynthetic molecules, such as, but not
limited to, plastoquinone and carotenoids. The photosynthetic
compounds of the present disclosure may be isolated from the host
organism and organelles in which they originate, or they may be
located in a thylakoid membrane or thylakoid organelle or
photosynthetic bacterial organism.
[0053] As used herein, the term "photosynthetic reaction center"
(PSRC) refers to one or more photosynthetic compounds as defined
above. A PSRC may include a single photosynthetic compound (e.g.,
PSII) or it may contain a group of photosynthetic compounds,
whether isolated or working in a cluster or entity (e.g., thylakoid
membrane or photosynthetic organism). A PSRC, as used in the
present disclosure, has the ability to harness light energy to
induce a photochemical reaction to oxidize water molecules and
generate electrons.
[0054] As used in the present disclosure, two materials are in
"electrochemical communication" when electrons generated by a
chemical reaction of one material (e.g., photosynthetic reaction
centers) can be transferred to and/or accepted by the other
material (e.g., nanostructured material and/or electrode).
[0055] "Direct electron transfer," as used in the present
application indicates that an electron can be transferred to an
electrode (e.g., anode) from the photosynthetic reaction center
(PSRC) that catalyzed the reaction that produced the electron, as
opposed to having to be transferred to the electrode by a separate
shuttle molecule (e.g., a redox mediator or redox shuttle). In the
present application, direct electron transfer includes the transfer
of electrons generated from a photosynthetic protein to the
electrode through a nanostructured material or matrix of
nanostructured materials, such as where the nanostructured material
couples the photosynthetic proteins, thylakoid membrane and/or
photosynthetic organism to the electrode. The presence of direct
electron transfer in an electrochemical cell of the present
disclosure does not preclude the existence of some electron
transfer occurring through a mediator, it just indicates that
direct electron transfer is occurring in the cell.
[0056] As used herein, the term "anode composite" refers to a
construct that provides the anode function in a photosynthetic
electrochemical cell of the present disclosure. Thus, the anode
composite includes the anode as well as any other materials or
components coupled to the anode that provide for the oxidizing
capability of the anode (e.g., nanostructured matrix material,
photosynthetic reaction centers, and the like, as well as compounds
or liking agents used to couple the anode to the other components
of the anode composite). Similarly, the term "cathode composite"
refers to a construct that includes the cathode as well as other
materials that provide for the reducing activity of the cathode
(e.g., the cathode and a compound capable of reducing O.sub.2, as
well as any compounds or agents used to couple the cathode to the
other components of the cathode composite, such as nanostructured
materials and/or any linking agents).
[0057] The term "matrix of nanostructured materials", as used in
the present disclosure, includes a network or multi-dimensional
structure of nanoparticles capable of coupling photosynthetic
proteins, a thylakoid membrane or organelle to an electrode.
[0058] "Redox mediator" or "redox shuttle" refers to a compound
capable of assisting in the transfer of electrons between a redox
enzyme (e.g., a photosynthetic compound of the present disclosure
that oxidizes water and generates electrons) and an electrode.
[0059] Having defined some of the terms herein, the various
embodiments of the disclosure will be described.
DESCRIPTION
[0060] Embodiments of the present disclosure include photosynthetic
electrochemical cells capable of generating an electric current
using light induced photo-electrochemical reactions catalyzed by
photosynthetic compounds. The present disclosure also includes
methods of generating an electrical current using photosynthetic
compounds, thylakoid membranes, and/or photosynthetic bacteria or
portions of photosynthetic bacteria to harness light energy and
using direct electrochemical communication to transfer electrons
generated by the photosynthetic proteins to an electrode.
[0061] In embodiments of the photosynthetic electrochemical cells
of the present disclosure, the cell includes an anode composite
that includes an anode (substrate electrode) and a photosynthetic
reaction center (catalyst) including one or more photosynthetic
compounds. The cell also includes a cathode or cathode composite
including a cathode (substrate electrode) and at least one enzyme
or metallic catalyst capable of reducing oxygen or other reductant.
In embodiments of the present disclosure, the anode composite
harnesses light energy to oxidize water molecules and generate
electrons for transfer to the cathode for reduction of oxygen.
Thus, the cathode uses electrons from the anode to reduce O.sub.2,
which induces a potential difference between the anode and the
cathode and generates an electrical current. The methods and the
photosynthetic electrochemical cells of the present disclosure,
therefore, provide a method of harnessing light energy to generate
an electrical current through photo-induced electrochemical
reactions and direct electron transfer.
[0062] In embodiments, the anode composite includes a
photosynthetic reaction center (PSRC), or multiple PSRCs. The PSRC
includes one or more photosynthetic compounds, and such
photosynthetic compounds can include proteins, pigments,
protein/pigment complexes, and other non-protein compounds involved
in photosynthesis. In embodiments of photosynthetic electrochemical
cells of the present disclosure, the PSRC of the anode composite
includes at least one photosynthetic protein capable of oxidizing
water molecules and generating electrons using light induced
photo-electrochemical reaction. The electrons produced by the PSRCs
are conducted to the anode via direct electron transfer. While not
every electron produced by the PSRC will necessarily be conducted
to the anode via direct electron transfer (some may be lost, and in
some embodiments, a portion of the electrons generated may be
transferred to the anode via a redox mediator), it will be
understood that at least a portion of the electrons are conducted
to the anode via direct electron transfer. In embodiments of the
photosynthetic electrochemical cells of the present disclosure, the
PSRC includes one or more of the following photosynthetic
compounds: PSII, PSI, plastoquinone, cyt b.sub.6f, plastocyanin,
phycocyanin, phycoerythrin, and carotenoids. The PSRC may include a
combination of the above photosynthetic compounds. In embodiments,
the PSRC of the electrochemical cell of the present disclosure
includes at least one photosynthetic protein or protein complex,
such as, but not limited to, PSI, PSII, cyt b.sub.6f, plastocyanin,
phycocyanin, and phycoerythrin. In embodiments, the PSRC can also
include non-protein photosynthetic compounds such as, but not
limited to, plastoquinone and carotenoids. In embodiments, the PSRC
may include one or more, two or more, three or more, or any
combination of the above photosynthetic compounds. In embodiments,
the anode composite may include one or more PSRCs where each PSRC
may include one or more photosynthetic compounds.
[0063] The photosynthetic proteins and compounds included in the
photosynthetic electrochemical cells of the present disclosure may
be isolated (e.g., removed from their natural environment,
organism, organelle, membrane, etc.) or they may be included in a
thylakoid membrane, an in-tact thylakoid organelle, a
photosynthetic organism (e.g. a photosynthetic bacterium) or a
photosynthetic portion of a photosynthetic organism (e.g., a
portion of the organism that is capable of photosynthesis when
isolated from the source organism).
[0064] In embodiments of the photosynthetic electrochemical cells
of the present disclosure, the PSRCs are included in a
photosynthetic entity coupled to the anode. In some embodiments
where the photosynthetic compounds are isolated from their natural
environment, they can be included in a synthetic photosynthetic
structure (e.g., a structure made of nanostructured materials, such
as the matrix of nanostructured materials described in greater
detail below). In other embodiments the photosynthetic compounds
are present in a natural photosynthetic entity (e.g., a thylakoid
organelle, a thylakoid membrane, a photosynthetic organism, or a
portion of a photosynthetic organism). Using the proteins in a
natural environment, such as a thylakoid membrane or photosynthetic
bacterium, may provide certain advantages, such as facilitating the
coordinated transfer of electrons between the various
photosynthetic compounds in the membrane/organism. This offers
various pathways for electron transfer between the photosynthetic
compounds in the PSRCs and the anode, rather than just a single
path offered by a single isolated photosynthetic protein. Thus, in
some embodiments it may be advantageous to utilize natural
photosynthetic entities, such as a portion of a thylakoid membrane,
an intact thylakoid organelle, or photosynthetic organism, or part
thereof in the electrochemical cells of the present disclosure.
[0065] As used herein, a PSRC can refer to an isolated
photosynthetic compound, to a grouping or cluster of photosynthetic
compounds working together, to a synthetic structure including a
cluster of photosynthetic compounds, to the photosynthetic
compounds or groups of compounds within such a synthetic structure,
to a single photosynthetic entity such as a thylakoid membrane,
thylakoid organelle or photosynthetic organism, or to a group of
photosynthetic compounds within such a photosynthetic entity. Thus,
a PSRC refers to any single or grouping of photosynthetic compounds
capable of oxidizing water molecules and generating electrons using
light induced photo-electrochemical reactions.
[0066] The photosynthetic compounds (whether isolated or part of a
natural or synthetic photosynthetic structure) are included in the
anode composite such that the PSRCs are in electrochemical
communication with the anode so that electrons generated during
photosynthetic reactions can be conducted directly to the anode. In
some embodiments, the PSRCs are coupled to the anode by a
nanostructured material. In such embodiments, the anode composite
also includes a nanostructured material in electrochemical
communication with at least one PSRC and the anode. In embodiments
where the PSRCS are isolated photosynthetic compounds or clusters
of isolated compounds, the PSRCs may be coupled to and/or
integrated into a nanostructured material such that they form a
synthetic photosynthetic structure, as described above, and the
photosynthetic structure can be coupled directly to the anode. In
other embodiments, the PSRCs may be a natural photosynthetic
entity, and the PSRC/entity may be coupled to the anode via a
matrix of nanostructured material. In some embodiments, the anode
can be functionalized with a nanostructured material, and the PSRC
is in electrochemical communication with the nanostructured
material of the anode.
[0067] In embodiments, the nanostructured material is a matrix of
nanostructured material made of a material capable of being coupled
to and in electrochemical communication with the PSRCs. In
embodiments, the nanostructured materials include, but are not
limited to, carbon based nanomaterials, metallic nanoparticles,
semiconductor nanoparticles, quantum dots or combinations of these
materials. Some embodiments of carbon based nanomaterials useful
for the electrochemical cells of the present disclosure include,
but are not limited to, materials such as carbon nanotubes,
multi-walled carbon nanotubes, fullerenes, carbon nanoparticles,
graphenes, two dimensional carbon nanosheets, graphite platelets,
and the like. In some specific embodiments, the matrix of
nanostructured material is multi-walled carbon nanotubes.
[0068] In the electrochemical cells of the present disclosure, in
embodiments, the PSRCs are coupled to the matrix of nanostructured
material in the anode by a cross-linking agent, such as, but not
limited to, 1-pyrenebutanoic acid succinimidyl ester (PBSE) or
other protein homo- or hetero-bifunctional cross-linking agent.
[0069] FIG. 1 provides an illustration of an embodiment of an anode
composite of the present disclosure, showing the electrode, the
matrix of nanostructured materials provided as multi-walled carbon
nanotubes (MWCNTs), and a thylakoid membrane as a photosynthetic
entity/PSRC including a combination of photosynthetic proteins and
other photosynthetic compounds in electrochemical communication
with the anode via the matrix of MWCNTs and PBSE likers.
[0070] Although the methods and photosynthetic electrochemical
cells of the present disclosure allow for direct electron transfer
between the PSRCs and the anode, in some instances it may be
advantageous to include a redox mediator (also known as a redox
shuttle) to facilitate transfer of electrons between the
photosynthetic proteins and the nanostructured material/anode. In
embodiments the redox mediator may be chosen from mediators such
as, but not limited to, ferricyanide, quinone-based compounds,
osmium complex based compounds, any other redox chemical compound
and combinations of the above.
[0071] In embodiments of the photosynthetic electrochemical cells
of the present disclosure, the cathode includes at least one
compound capable of reducing a reductant, such as, but not limited
to O.sub.2, ferro/ferricyanide couple, and the like. In
embodiments, the photosynthetic electrochemical cells of the
present disclosure include a cathode composite including a cathode
and a compound or combination of compounds capable of reducing a
reductant, such as O.sub.2. In embodiments, the cathode composite
may also include a nanostructured material to facilitate
electrochemical communication between the cathode and the
oxygen-reducing compounds. The nanostructured material of the
cathode composite can be any of the nanostructured materials
described above in reference to the anode composite. In
embodiments, the compound capable of reducing O.sub.2 can include,
but is not limited to, an enzyme or a metallic catalyst. In some
embodiments the enzyme capable of reducing O.sub.2 can include, but
is not limited to, laccase, bilirubin oxidase, ascorbate oxidase,
tyrosinase, catechol oxidase, and combinations of these or other
enzymes. In embodiments the enzyme capable of reducing O.sub.2
include a metallic catalysts such as, but not limited to platinum,
silver, gold, cobalt, nickel, iron and combinations of these
metals. In embodiments, the compound capable of reducing
ferro/ferricyanide couple, can include but is not limited to, could
be a metal, semiconductor or carbon or a chemical capable of
reducing the ferro/ferricyanide couple.
[0072] In embodiments of the present disclosure the anode and
cathode can be made of any standard electrode material, such as
carbon, metals, semiconductor such as silicon, and the like. One
advantage to the cells of the present disclosure is that the use of
the nanostructured materials to couple the photosynthetic compounds
or oxygen reducing compounds to the electrodes allows coupling of
the photosynthetic compounds to the electrode without the need for
expensive precious metal electrodes (e.g., gold, silver, platinum,
etc.) and without complex immobilization procedures that are
incompatible with other, less expensive electrode materials such as
carbon.
[0073] The examples below and the attached figures provide
additional detail regarding some embodiments of the photosynthetic
electrochemical cells of the present disclosure and the elements of
the anode and cathode described above. FIG. 7A provides an
illustration of an embodiment of an electrochemical cell of the
present disclosure.
[0074] The methods of the present disclosure include methods of
generating an electrical current including using the photosynthetic
electrochemical cells of the present disclosure to convert light
energy to electrical energy. In embodiments, the methods of the
present disclosure include using thylakoid membrane photosynthetic
proteins and compounds and/or photosynthetic bacterial proteins and
compounds to harness light energy to oxidize a water molecule and
directly transfer electrons from the photosynthetic proteins to an
anode using direct electrochemical communication. In embodiments,
methods of the present disclosure also include using the electrons
from the anode to reduce O.sub.2 at a cathode, thereby inducing a
potential difference between the anode and the cathode and
generating an electrical current.
[0075] Briefly described, some methods of generating an electrical
current according to the present disclosure include providing an
electrochemical cell that has an anode composite that includes a
PSRC in electrical communication with an anode via a nanostructured
material and a cathode composite capable of reducing oxygen. The
electrochemical cell is exposed to light in the presence of water
so that the PSRC uses light energy to oxidize water molecules and
generate electrons, which are transferred to the anode via the
nanostructured material, flow to the cathode, where they reduce
oxygen, thereby inducing a potential difference between the anode
and the cathode to generate an electrical current.
[0076] In embodiments the present disclosure includes methods of
generating an electrical current by providing a photosynthetic
electrochemical cell of the present disclosure and exposing the
photosynthetic electrochemical cell to light in the presence of
water, such that the PSRCs use light energy to oxidize a water
molecule and generate electrons which are transferred to the anode
via the nanostructured material, and the electrons generated at the
anode flow to the cathode where they are used to reduce O.sub.2 at
a cathode, thereby inducing a potential difference between the
anode and the cathode and generating an electrical current. The
photosynthetic electrochemical cells used in the methods of the
present disclosure can include an anode composite having
photosynthetic reactions centers in electrical communication with
an anode via a nanostructured material as described above and
include a cathode or cathode composite capable of reducing O.sub.2
or other reductant as described above.
[0077] Now having described the embodiments of the present
disclosure, in general, the Examples, below, describe some
additional embodiments of the present disclosure. While embodiments
of the present disclosure are described in connection with the
Examples and the corresponding text and figures, there is no intent
to limit embodiments of the present disclosure to these
descriptions. On the contrary, the intent is to cover all
alternatives, modifications, and equivalents included within the
spirit and scope of embodiments of the present disclosure.
EXAMPLES
Example 1
Manipulating Electron Transport Pathways in Thylakoid Composites
for Photosynthetic Energy Conversion
[0078] Spinach thylakoids were coupled to electrodes via
multiwalled carbon nanotubes using a molecular tethering chemistry.
The resulting thylakoid-carbon nanotube composite showed high photo
electrochemical activity under illumination. It is believed to be
the first time multiple membrane proteins have been observed to
participate in direct electron transfer with the electrode,
resulting in the generation of photocurrents. Thus, it is believed
that the present disclosure describes the first of its kind for
natural photosynthetic systems.
[0079] The high electrochemical activity of the thylakoid-MWNT
composites has significant implications for both photosynthetic
energy conversion and photofuel production applications. A fuel
cell type photosynthetic electrochemical cell developed using
thylakoid-MWNT composite anode and laccase cathode produced a
maximum power density of 5.3 .mu.W cm.sup.-2 comparable to that of
enzymatic fuel cells. The carbon based nanostructured electrode has
the potential to serve as an excellent immobilization support for
photosynthetic electrochemistry based on the molecular tethering
approach as demonstrated in the present example.
Introduction
[0080] Plant photosynthesis has evolved over 2.5 billion years to
convert solar energy into chemical energy using only water, with an
unmatched quantum efficiency of nearly 100%.sup.(1,2). In recent
years, there has been an increasing interest in mimicking the
natural photosynthetic process for energy conversion and photo fuel
(ethanol, H.sub.2 etc.) production.sup.(3-5). This is being carried
out using synthetic routes such as metal oxides, semiconductors or
chemical catalysts for carrying out the light-driven water
splitting reaction.sup.(6-11). Alternatively, components of the
naturally occurring photosynthetic apparatus of bacteria.sup.(12),
algae and plants.sup.(13-16) have been employed for bioconversion
applications. For example, the direct conversion of light into
electricity based on photosynthesis in an electrochemical cell has
been investigated in the past.sup.(17-20), using natural systems
such as thylakoids, chlorophyll molecules, photosynthetic reaction
centers.sup.(21-28), and even whole cells such as
cyanobacteria.sup.(29-31). Besides these representative attempts,
the low electron transfer efficiency of the photosynthetic
machinery to the electrodes still plague the power output
performance of these systems. Isolated photosynthetic components
systems possess some advantages over whole cells, such not needing
nutrients for sustenance, and not having competition between
respiration and photosynthesis in sharing the electron transfer
pathways. However when isolated plant photosynthetic systems have
been used on electrodes they have suffered from low efficiencies
due biomolecule stability, improper immobilization, lack of
electrical communication, etc.sup.(10). For light energy harvesting
applications, it is thermodynamically advantageous to collect
electrons directly from the molecules at high-energy states along
the photosynthetic electron transport pathway, such as an excited
photosystem II (PSII).sup.(26,32,33). Moreover, for direct
light-electricity conversion applications, it is generally
preferable to use a higher order plant based system that uses only
water as the electron donor such as PSII, rather than isolated PSI
complexes, which require an alternate electron donor. For this
purpose, attempts have been made to immobilize PSII reaction
centers on to the electrode using cytochromes.sup.(34) or
nickel-nitrilotriacetic acid.sup.(35) as cross-linkers or through
some terminal electron acceptors such as Co.sup.III
complexes.sup.(36). All these methods use precious metals (e.g.,
gold) as the immobilization support and use expensive
immobilization procedures that cannot be extended to other
electrode materials (e.g., carbon). Accordingly, the precious metal
based electrochemistry carries less practical value for energy
conversion applications.
[0081] On the other hand, photosynthetic organelles or membranes
also possess advantages over isolated reaction center complexes for
electrochemical applications. Some such advantages include: high
individual protein stability.sup.(37), the ability to use simpler
immobilization procedures, and the presence of multiple electron
transfer routes. For example, if thylakoid membranes are used in
the place of isolated PSII complex, then the electron transfer from
an oxygen evolving complex (OEC) site to the electrode can be
achieved via plastoquinone, cytochrome (cyt) b.sub.6f,
plastocyanin, ferredoxin, PSI, etc. in addition to a direct
transfer from PSII.sup.(34). Moreover retention of their natural
partners results in enhanced stability of the individual proteins
in thylakoids in comparison to their isolated
counterparts.sup.(38). Therefore using thylakoids as
photo-biocatalysts or otherwise complexing photosynthetic proteins
within a photosynthetic structure offers the potential for high
photo-electrochemical activity as well as high stability for both
energy conversion and fuel production applications.
[0082] The present example demonstrates the photo-electrochemical
activity of spinach thylakoids immobilized on to multi-walled
carbon nanotube (MWNT) modified electrodes. By employing a
carbon-based material, the necessity for expensive precious
metal-based catalyst supports to immobilize photosynthetic
machinery onto electrode surfaces was eliminated. The molecular
tethering approach described in the present example by using
nanostructured materials to couple the photosynthetic structure to
the electrode helps establish multiple attachments between the
thylakoid membranes and the electrode surface. Moreover, the
present example demonstrates that by using the entire membrane
instead of isolated photosystem complexes, manipulation of the
electron transfer pathways to achieve high electron transfer flux
for photo current generation was possible. This example also
demonstrates direct light to energy conversion with water as the
only input using a photosynthetic fuel cell composed of a thylakoid
based anode and laccase based cathode, first of its kind employing
a plant photosynthetic membrane and an enzymatic cathode operating
at neutral pH. A schematic of the thylakoid membranes immobilized
on MWNT modified electrode and the associated electron transport
pathways are shown conceptually in FIG. 1.
EXPERIMENTAL
Materials
[0083] Thylakoids were extracted from fresh organic spinach
obtained from local market. MWNT, 10 nm diameter and 1-2 .mu.m
length (Dropsens, Spain) was used as the immobilization support for
the thylakoids. 1-pyrenebutanoic acid succinimidyl ester (PBSE)
(Anaspec) was used as the molecular tethering agent to attach
thylakoids on MWNT. Potassium ferricyanide, redox mediator, and
N,N-dimethyl formamide (DMF), solvent used for reagent preparation,
were purchased from Acros Organics.
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was purchased from
Tokyo Chemical Industry. Laccase from Trametes versicolor (Sigma)
was the enzyme used on the cathode. Potassium cyanide (KCN) was
purchased from Fisher Scientific. Paraquat/Diquat was purchased
from Ultra Scientific. Tricine (OmniPur), sorbitol (EMD Chemicals
Inc), ethylenediaminetetraacetic acid (EDTA) (VWR), and potassium
hydroxide (Mallinckrodt Baker) were used for preparing buffer
solutions. Phosphate buffer for electrochemical testing was
prepared using monobasic and dibasic potassium phosphates (VWR).
All buffers were prepared using nanopure distilled water
(ddH.sub.2O). Electrolyte (buffer) solutions were purged for 30 min
with N.sub.2 to remove any dissolved O.sub.2.
Methods
[0084] Thylakoids were isolated from Spinacia oleracea (spinach)
leaves based on the procedure given in literature.sup.(39) using a
refrigerated centrifuge (Beckman Coulter Avanti J-E). The procedure
is known to produce a mixture of both intact organelles and broken
thylakoid membranes. During isolation the chlorophyll concentration
was determined to be between 2.5 and 3.0 mg mL.sup.-1 (average of
2.8 mg mL.sup.-1) via UV-Vis absorbance measurements using a
spectrophotometer (UV-Vis) (Cary Varian 50 Bio, Sparta, N.J.) using
the formula given in the FIG. 8. The oxygen evolution activity of
isolated thylakoids was measured in oxygen deprived tricine buffer
pH 7.8 with a standard Clark O.sub.2 electrode. The isolated
thylakoids were in the form of a pellet and was stored in the dark
at -80.degree. C. The pellets were re-suspended in the buffer when
needed for electrode preparation. For control experiments, the DCMU
exposed thylakoids were prepared by suspending thylakoids in 0.1 mM
DCMU solution and incubated in ice bath for 30 min. Similarly in
another control experiment, 250 .mu.g mL.sup.-1 solution of
paraquat/diquat solution was used to suspend thylakoids and
incubated in ice bath for 30 min.
[0085] Slurries of MWNT were prepared by dispersing 1 mg mL.sup.-1
of MWNTs in 10 mM DMF by 10 min ultra sonication using an
ultrasonic homogenizer (Omni International) at the power output of
20 watts. The dispersion was sonicated again for 1 h in a bath
sonic cleaner (XP-Pro). The obtained MWNT dispersion was used as it
is for electrode modifications.
[0086] A molecular tethering approach was used to immobilize
thylakoid membranes on the carbon nanotube matrix using PBSE as the
linker (e.g., tethering agent), which has been demonstrated to
produce excellent bio-electrochemical characteristics.sup.(40-42).
In this method, first the electrodes (0.02 cm.sup.2) were polished
with 0.05 .mu.m alumina slurry. The polished electrode was rinsed
and ultrasonicated in ddH.sub.2O for 8 min. Then the electrode was
modified with 4 .mu.L of MWNT dispersion and later dried at
70.degree. C. After drying, a desired volume of 10 .mu.M PBSE was
drop casted on the MWNT modified electrode and incubated for 15 min
in an ice bath. The resulting modified electrode was washed first
with DMF to remove the loosely bounded PBSE and then with tricine
buffer to neutralize the pH of the electrode surface. Finally, 5
.mu.L of thylakoid suspended solution (corresponds to 0.44 .mu.g
cm.sup.-2 chlorophyll loading) was drop casted on the electrode
surface and incubated for 1 h in the dark in an ice bath. The
modified electrode was then washed with tricine buffer prior to
experimentation.
Testing
[0087] Bare or thylakoid modified MWNT was used as the working
electrode in a 3-electrode electrochemical cell setup with a
platinum wire counter electrode (Alfa Aesar) and a silver-silver
chloride (Ag/AgCl) reference electrode (CH Instruments). All
electrochemical experiments were conducted at 25.+-.2.degree. C.
using 0.1 M tricine buffer pH 7.8 as the electrolyte.
Electrochemical tests were performed both in the presence and
absence of [Fe(CN).sub.6].sup.3-/4- as a redox mediator. The
operating conditions for electrochemical tests were chosen based on
a series of optimization tests for thylakoid loading,
immobilization duration, mediator concentration and anode potential
over a reasonable range, the results of which are given in FIGS.
9A-9B. The O.sub.2 evolution activity during experiments was
monitored via a Clark-type O.sub.2 electrode (VWR Symphony
Dissolved Oxygen Probe). Cyclic voltammetry (CV), current vs. time
(i-t curve, steady state current) and electrochemical impedance
spectroscopy studies were conducted using CHI-920c model
potentiostat (CH Instruments). A Dolan-Jenner Industries Fiber-Lite
model 190 lamp was used for light illumination with `high`
intensity setting of 80 mW cm.sup.-2. Surface morphology of the
immobilized thylakoids was studied using a scanning electron
microscope (SEM) (FEI Inspect F FEG-SEM), atomic force microscope
(AFM) (Veeco Multimode Nanoscope). Absorption spectra were obtained
using Genesys 10S UV-Vis spectrophotometer (Thermo Scientific). The
photosynthetic fuel cell was constructed using thylakoid-MWNT
composite modified anode and laccase-MWNT composite modified
cathode. The electrodes were held inside a glass vial containing
0.1 M phosphate buffer solution (pH 6.8) as electrolyte. No oxygen
was bubbled during the experiment and the oxygen available in the
electrolyte was reduced to water at the cathode.
Chlorophyll Concentration Measurements
[0088] The chlorophyll concentration (Cch) was calculated by using
the data from UV-V is spectrum into the equation (E1).
C ch ( mg mL ) = 8.02 .times. A 663 + 20.2 .times. A 645 10
.fwdarw. ( E1 ) ##EQU00001##
Optimization of Composite Composition
[0089] The plots of current versus time at fixed anode potentials
were used as a guiding tool for optimizing the thylakoid-MWNT
composite electrode (FIGS. 9A-D). The parameters optimized were
thylakoid immobilization time (FIG. 9A) and thylakoid concentration
in the immobilization mixture (FIG. 9B), mediator concentration
(FIG. 9C), and the anode polarization potential (FIG. 9D). The
results showed the photocurrent was directly proportional to
thylakoid concentration; however, only a maximum of 0.44 .mu.g
cm.sup.-2 of thylakoids could be immobilized due to the limited
geometric size of the electrodes being used in the experiment.
Similarly, it was noticed that incubation durations (for
immobilization of thylakoids on MWNT matrix) beyond 1 h did not
result in a significant increase in photocurrents. The photocurrent
was also proportional to the mediator concentration, but the
percent decrease of photocurrent per duty cycle varied. The
optimized mediator concentration obtained was 1.5 mM, where the
photocurrent was 0.9 .mu.A for the first cycle while still
maintaining stability through multiple cycles. The anode potential
was also optimized at 0.2 V to observe noticeable
photo-activity.
Results and Discussion
Physical Characterization
[0090] Tapping mode AFM and SEM were used to study the morphology
of thylakoid-modified MWNT electrodes. The AFM topography,
amplitude and phase images of the unmodified MWNT (control) and
thylakoid-modified MWNT electrodes are shown in FIG. 2. The
unmodified control electrode with the bare MWNT matrix shows clear
and identifiable nanotubes as seen in FIG. 2A. The thylakoid
modified-MWNT composite matrix shown in FIG. 2B has a similar
morphology as that of the control, but with distinct thylakoids on
the surface. Due to the blanketing effect of biological structures,
the underlying MWNT fibrils in the composite are not obvious in the
topography image, but are evident in amplitude and phase images in
FIG. 2B. The presence of thylakoids is shown by the lighter
extruding region in the composite (FIG. 2B), sized approximately 3
.mu.m.+-.0.5 in length which agrees with the typical size of a
thylakoid unit.sup.(43). A thylakoid modified electrode surface in
the absence of MWNT is also shown in FIG. 2C for comparison and
verification. The surface roughness of the thylakoid matrix is 9
nm.+-.0.5, which is close to the roughness of a monolayer. The
roughness values are similar for both thylakoids (FIG. 2C) and
thylakoid-MWNT composite matrices (FIG. 2B). The surface coverage
of active membrane proteins of thylakoids on the electrode surface
is discussed in section 3.3.1. The morphology studied using SEM
also show the length of thylakoids to be 3 .mu.m.+-.1 as shown in
FIG. 3. This is in good agreement with the AFM observations.
Electron Transfer Pathways in Thylakoids
[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 generated as a
result of the photo-induced water oxidation 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): PSII.fwdarw.plastoquinone.fwdarw.cyt
b6f.fwdarw.MWNT.fwdarw.electrode. ETP1 is possible if the cyt b6f
is adsorbed or molecularly tethered to MWNT surface and its redox
site placed closely to MWNT to enable direct electron transfer. The
second possible electron transfer pathway (ETP2) is depicted by
arrows (b): PSII.fwdarw.plastoquinone.fwdarw.cyt
b6f.fwdarw.plastocyanin.fwdarw.MWNT.fwdarw.electrode. Here the
electron gets routed to plastocyanin before reaching the MWNT
matrix. Since plastocyanin may freely diffuse between the stromal
and lumenal sides of a ruptured thylakoid membrane (as in the case
of our experiments) it is likely to participate in the electron
conduction as well. Both ETP1 and ETP2 serve as direct electron
transfer routes for electrochemical charge transfer. Upon the
addition of a mediator such as [Fe(CN).sub.6].sup.3-/4-, a third
pathway as indicated by arrows (c) is also possible for electron
conduction to the electrode (ETP3):
PSII.fwdarw.plastoquinone.fwdarw.cyt
b6f/plastocyanin.fwdarw.[Fe(CN).sub.6].sup.3-/4-.fwdarw.MWNT.fwdarw.elect-
rode. In this case, ferricyanide mediates the electron transfer
from multiple membrane proteins in the thylakoid to the MWNT
electrode. Therefore the lack of electrical connectivity between
those membrane proteins and electrode is not of a concern.
[0092] In addition to the three ETPs discussed above, other routes
for electrochemical communication between thylakoids and MWNT
electrode are possible. For example, a direct electron transfer
from plastoquinone site of PSII to MWNT is possible if the stromal
side of PSII complex is orientated towards the electrode. However
the present experimental results (discussed in the following
paragraphs) indicated no significant contribution from any
additional routes to the electrochemical charge transfer and hence
were not depicted in FIG. 1 or explored in detail. Also, since the
primary focus of this example is on the electrons generated at PSII
complex by water splitting reaction, other ETPs originating from
PSI.sup.(44) or from electrolyte impurities.sup.(45) are not
discussed here.
Redox Electrochemistry of Immobilized Thylakoid Membranes
[0093] Direct Electron Transport
[0094] Cyclic voltammetry was used to study redox activity of the
unmodified and thylakoid-MWNT composite modified electrodes and to
verify the existence of the ETPs discussed above in FIG. 1. The
electrodes were cycled between -0.7 to 0.5 V vs. Ag/AgCl at a scan
rate of 0.02 V s.sup.-1. FIG. 4A compares the cyclic voltammograms
of thylakoid-MWNT composites in the presence and absence of
[Fe(CN).sub.6].sup.3-/4- as mediator. The formal potential
(E.sup.0) values for peaks 1.sub.peak and 3.sub.peak as observed in
our results were at .about.0.035 and .about.0.2 V (see FIG. 4A),
which fell closely with that of the redox potentials of cyt
b.sub.6f (Fe.sup.II/III) and plastocyanin (Cu.sup.I/II),
respectively, when the pH difference was accounted.sup.(46-48). No
redox peak directly attributable to plastoquinone was observed in
our experiments, which is located inside the thylakoid lipid
bilayer. The surface coverage of the cyt b.sub.6f and plastocyanin
was 1.2.times.10.sup.-9 and 0.6.times.10.sup.-9 mol cm.sup.-2
respectively. These values were calculated using the equation
.GAMMA.=Q/nFA, where .GAMMA. is surface coverage, Q is the charge
measured from the cyclic voltammogram's cathodic peak, n is the
number of electrons, F is the Faraday constant, and A is the
geometric area of the electrode. Since thylakoids may exist as
broken membrane particles instead of intact organelles in our
samples, the observance of redox peaks for a lumen-side soluble
protein such as plastocyanin is not surprising. The cyt b.sub.6f
and plastocyanin redox centers are much smaller than other reaction
center complexes such as PSII and PSI. Therefore, naturally the
MWNTs have easier access to cyt b.sub.6f and plastocyanin for a
direct electrochemical charge transfer resulting in distinct redox
peaks as observed in FIG. 4A. To confirm that the peaks at 0.2 V
can be attributed to plastocyanin redox activity, a separate
plastocyanin-inhibition experiment was conducted to study the
effect of inhibition on this redox peak. For this purpose KCN (10
mM) was added as an inhibitor to the thylakoid solution prior to
immobilization. KCN inhibits the plastocyanin activity in
photosynthesis by blocking its CuI/II active site at high
concentrations (>10 mM) and high pH values (>7.5).sup.(49).
The voltammograms of thylakoid modified MWNT electrode with KCN
inhibitor showed a dramatic 73% reduction in the oxidation peak at
0.2 V as shown in supplementary FIG. 10. This supports the
conclusion that the redox response in the 0.19 V-0.2 V range was
that of plastocyanin in thylakoid modified electrodes. This is one
of the few reports that demonstrate a direct electrochemistry for
thylakoid-based electrodes.
Mediated Electron Transport
[0095] In a separate set of experiments, 1.5 mM
[Fe(CN).sub.6].sup.3-/4- couple was added to the electrolyte to
assist electron transfer from thylakoid membrane to the MWNT
electrode. Ferricyanide is a suitable choice because of its minimal
photo-activity.sup.(50,51), compared to benzoquinone mediators used
elsewhere.sup.(52), as confirmed in separate studies (see FIG. 11
for relevant data). As shown in FIG. 4A, in the presence of
[Fe(CN).sub.6].sup.3-/4-, a single dominant oxidation peak
(2.sub.peak at E.sup.0 0.16 V) was observed masking the redox
responses of both cyt b6f and plastocyanin observed. This confirms
the existence of mediated electron transport pathway (ETP3) as
suggested in FIG. 1. This also indicates that the electron flux due
to mediated transport was much higher than that of direct transport
through the membrane bound proteins. The O.sub.2 evolution due to
photo-induced water splitting in thylakoid modified MWNT electrodes
upon illumination is also evident in the voltammograms of FIG. 4B.
The oxygen reduction and ferricyanide oxidation were observed at
E.sub.pc=-0.4 V and E.sub.pa=0.3 V vs. Ag/AgCl respectively in FIG.
4B. Upon illumination the peak currents for their reactions
increased by 1.0 (I.sub.pa) and 0.2 (I.sub.pc) .mu.A respectively
indicative of photo-catalysis by immobilized thylakoids. The rate
of oxygen evolution from the photo-induced water oxidation was
measured to be 253 .mu.mol O2 mg chl.sup.-1h.sup.-1 (using a Clark
electrode) indicating high photo-activity of the reactions centers
complexes in thylakoids. The results suggest that the electron flux
to electrode could be greatly enhanced by [Fe(CN).sub.6].sup.3-/4-,
which acts as mediator for electrochemical charge transfer between
thylakoid and MWNT electrode. To ensure that the mediator did not
interfere with light-absorbance activity of thylakoids, individual
absorption spectra were obtained for both thylakoids (in 80%
acetone solution) and [Fe(CN).sub.6].sup.3-/4- couple using UV-Vis
spectroscopy. As shown in FIG. 12A, the absorption spectrum for
[Fe(CN).sub.6].sup.3-/4- mediator did not show any absorbance at
673 nm, the wavelength at which chlorophyll-a absorbs light in
PSII. Also the presence of mediator did not hinder the light
absorbance of chlorophyll-a molecule in our thylakoid-MWNT
composites (see FIG. 12A for relevant data).
Photo-Electrochemical Activity of Thylakoid-MWNT Composites
[0096] The photo-electrochemical activity of thylakoid modified
MWNT electrodes were evaluated using open circuit potential (OCP),
potentiostatic polarization, and AC impedance measurements.
Open Circuit Potentials
[0097] FIG. 5A compares the open circuit potentials (OCPs) of
unmodified and thylakoid modified MWNT electrodes in the presence
of mediator. Within 100 seconds after the addition of mediator
under dark condition, the OCP of the unmodified and thylakoid
modified MWNT electrodes stabilized at 0.23 and 0.19 V,
respectively. The slightly lower OCP for thylakoid-MWNT electrode
could be dictated by the mixed potential caused by a variety of
thylakoid membrane proteins whose individual redox potentials range
from -1.3 to +1.0 V vs. SHE.sup.46. Upon changing the illumination
conditions between light and dark over several light on-light off
cycles, a clear variation in the open circuit potential was
observed, indicative of photo-electrochemical activity for the
thylakoid-MWNT composite electrode. The variation was as high as 90
mV during the first on-off cycle, which eventually subsided during
subsequent cycles consistent with the attainment of dynamic
equilibrium over time. No such variation in OCP between light and
dark was observed for the unmodified MWNT electrode (control) in
FIG. 5A.
Potentiostatic Polarization
[0098] The photo-electrochemical activity of the thylakoid-MWNT
composite was evaluated at constant anode potential of 0.2 V and
the variation of anode current with time was evaluated during light
on-light off cycles. The photocurrent densities ranged from 30 to
70 .mu.A cm.sup.-2 during initial cycles eventually attaining
steady state within .apprxeq.400 s in the range of 23 to 38 .mu.A
cm.sup.-2 that lasted for 1 week. (FIG. 13). Table 1 compares the
photo-current densities observed in this example with that of other
photosynthetic systems reported in the literature and shows that
the anodic photocurrents observed here are about two orders of
magnitude larger than the highest values reported in the literature
for natural plant systems, either PSII based energy
conversion.sup.(52-54) or PSI based bio-hydrogen
production.sup.(55).
TABLE-US-00001 TABLE 1 Comparison of various photosynthetic anode's
photocurrent density. Electrode material Photocurrent/area .sup.b
Reference Au-MWNT-Thylakoid 68 .mu.A cm.sup.-2 This work
Au-poly(mercapto-p- 3.1 .mu.A cm.sup.-2 52 benzoquinone) Au
nanoparticles-PSII 2.4 .mu.A cm.sup.-2 53 Au-Thylakoid 0.2 .mu.A
cm.sup.-2 60 Au-PSII 0.1 .mu.A cm.sup.-2 54 Au-Thylakoid 1.1 .mu.A
cm.sup.-2 61 Au SAM-RC-RBS .sup.a 0.2 .mu.A 34 Carbon coated Au-
0.05 .mu.A 62 RC-RBS .sup.a SAM--self assembled monolayer;
RC--reaction center; RBS--Rhodobacter sphaeroides .sup.b
Photocurrent density was calculated using the photocurrent and the
active surface area provided in the respective literature
[0099] The thylakoid-MWNT composite was also found to be very
responsive to the light intensity (see FIG. 13 for light intensity
data). No significant photo-electrochemical activity was noticed
for the unmodified MWNT electrode without thylakoids or for
thylakoids physically adsorbed onto the MWNT without the tethering
cross-linker. The small photo-currents seen in the case of
unmodified MWNT in FIG. 4B, was primarily due to the light
absorbance of Fe(CN).sub.6].sup.3-/4- in the 410-420 nm range which
did not interfere with the light absorbance of chlorophyll in
thylakoids in the 660-675 nm range (as shown in the absorption
spectrum in supplementary FIG. 12A). In order to understand if
immobilized thylakoids have significant advantages over
freestanding suspended thylakoids in solution, separate
polarization experiments were performed using thylakoids suspended
in the electrolyte without immobilization on MWNT electrodes. For
such electrodes the photo-electrochemical activity was drastically
reduced (as shown in FIGS. 15A and 15B), indicating that the
composite based immobilization methods are best suited to establish
high photo-electrochemical activity in natural systems.
[0100] The decrease in the amplitude of photo-currents over
continuous light on-off cycles observed in FIG. 5B could be due to
a combination of two effects: (i) transience in mediator diffusion
between thylakoid and electrode before the attainment of a steady
state; (ii) photo-damage of proteins under extended light
exposure.sup.(56). The contribution of the first effect was studied
and confirmed in a separate set of experiments where the
thylakoid-MWNT composite electrodes were tested under different
duty cycles (different ratios of Time.sub.on/Time.sub.off) as well
as under continuous light on and continuous light off (dark)
conditions (see supplementary FIGS. 14A-14B for relevant data). The
second effect namely photo-damage (photoinhibition) occurs under
continuous and extended illumination conditions.sup.(57) and is an
inherent property of natural systems. In plants, natural biological
mechanisms repair the photo-damaged proteins.sup.(58).
AC Impedance
[0101] AC impedance studies were also carried out on thylakoid-MWNT
composites under light and dark conditions in the presence of
mediator to understand the influence of individual resistances on
photocurrent generation. The Nyquist plots (-Z'' vs. Z') of the
impedance data and the equivalent electrical circuit (to which the
data was fitted for parametric analysis) are given in FIG. 5C.
R.sub.s represent electrolyte resistance, R.sub.ct is charge
transfer resistance, CPE is a constant phase element between the
electrolyte solution and the modified electrode, and lastly Z.sub.w
represents the Warburg impedance due to the diffusion of mediator.
The shape of the Nyquist profiles shows a clear difference in the
impedance between light and dark condition, with the composites
showing lower impedance for charge transfer under the illuminated
condition. The fitted values for R.sub.ct were 87 and 317 k.OMEGA.
under light and dark, respectively. The results indicate an
enhanced kinetic activity due to lowered charge-transfer impedance
for electrochemical charge transfer in thylakoid-MWNT composites
under illumination. Though a Warburg element was included in the
equivalent circuit to ensure completeness in the representation of
the system, the diffusion limitations were not observed to play a
major role in this kinetic limited system, as can be noticed in the
shape of the Nyquist plots in FIG. 5C, where there was no
45.degree. Warburg slope. At the same time, an equivalent circuit
without the Warburg component did not result in the perfect fit of
the equivalent circuit data indicating that Warburg and kinetic
impedances (R.sub.et) were expressed with similar time
constants.
[0102] An impedance observation in the absence of mediator also
showed high charge transfer impedance indicating this system was
not limited by the mediator diffusion. Separate measurements
performed on thylakoid modified electrodes with and without MWNT
platform indicated lower ohmic resistance (after eliminating the
solution impedance contribution) for the MWNT electrodes. This
confirmed the existence of high electronic conductivity for the
thylakoid-MWNT composite electrodes.
Role of PSII Vs. PSI in Photocurrent Generation
[0103] In order to confirm that the light-induced water splitting
reaction is the electron source for the observed photocurrents in
our composite electrodes, two control experiments were performed.
The first control experiment was aimed at studying the effect of
blocking the PSII reaction center complex and the second was aimed
at blocking the PSI reaction center complex from participating in
the photosynthetic electron transport. For inhibiting PSII
activity, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added
to the thylakoid solution prior to immobilization. DCMU is a
herbicide that specifically blocks the Q.sub.A.fwdarw.Q.sub.B site
in PSII complex, severing the electron transport from PSII to the
subsequent proteins in the pathway.sup.(59). As shown in FIG. 6,
potentiostatic tests at 0.2 V on thylakoid-MWNT composites showed
that when DCMU was used, the thylakoid activity was severely
inhibited thereby drastically reducing the photocurrents during the
light on-off tests. This confirms that the electrons originate from
PSII. Interestingly the cyclic voltammograms thylakoid-MWNT
composites (FIGS. 19A-19B) showed retention of redox
electro-activity by both cyt b.sub.6f and plastocyanin even when
PSII was inhibited by DCMU. This suggests that blocking the PSII
complex with DCMU did not significantly affect the redox activities
of the other thylakoid membrane proteins individually, an
indication that the direct electron properties of thylakoid can be
utilized for electrochemical charge transfer as suggested above.
The inhibition of PSI (as shown in supplementary FIG. 17) reveals
that the presence of the PSI inhibitor did not significantly reduce
the photo-electrochemical response of the thylakoid-MWNT
composites. The results reiterate that water oxidation at PSII was
the major source for photocurrents in the thylakoid composites and
contribution of PSI complex to photocurrent generation was
insignificant in the present study. This also confirms that the
major routes for electron conduction from OEC site to the electrode
were the three ETPs proposed in FIG. 1, which presumes that all the
membrane integral proteins in thylakoid membrane are intact and
participate in photosynthetic linear electron transport.
Thylakoid-Laccase Photosynthetic Electrochemical Cell
[0104] A fuel cell type electrochemical cell was constructed using
the thylakoid-MWNT composite anode and laccase-MWNT composite
cathode and tested in an electrolyte solution (PBS buffer pH 6.8).
The anode oxidizes water upon illumination with light using
thylakoid-MWNT composites as photo-biocatalysts, whereas the
cathode reduces oxygen to regenerate water in the system using
laccase as an enzymatic bio-electrocatalyst. The use of laccase for
bio-electrocatalytic oxygen reduction in biological fuel cells has
been well established.sup.(40-42). The molecular tethering approach
used for thylakoid immobilization was also used for laccase
immobilization on MWNT at the cathode. The open circuit potential
of cell was .about.0.4 V. Polarization tests were performed under
illumination, at constant applied potentials between 0.35 V and 0 V
and the resulting steady state currents were measured and shown in
FIG. 7. The polarization curves showed a maximum current density of
.about.70 .mu.A/cm.sup.2 and a maximum power density of 5.3
.mu.W/cm.sup.2. The power density values were comparable to the
ones reported for PSII electrochemical cell using
gold-poly(mercapto-p-benzoquinone) anode and bilirubin oxidase
cathode.sup.(52). It is worthy to point out that our system was not
optimized for power density in any way. Rather this work was a
simple demonstration of power generation using thylakoid membrane
and laccase cathode. Further enhancements in current and power
densities can be achieved by reducing the anode-cathode separation
distance (to lower ohmic impedance), increasing the loading of
thylakoids and laccase onto the electrodes (to enhance
electro-kinetics) and by managing the mediator diffusion (to
minimize mass transfer limitation) for achieving high
performance.
Inhibition of Plastocyanin by KCN:
[0105] The thylakoid-MWNT composites prepared using KCN in the
immobilization mixture exhibited a significant reduction the
plastocyanin activity by up to 73%. Although KCN only inhibits
plastocyanin, the redox activity of cyt b.sub.6f (peak at 0 V) was
also reduced by 23%. This could be due to the lack of an electron
acceptor for cyt b.sub.6f (when plastocyanin was inhibited), which
may result in an excited cyt b.sub.6f (electron rich) that reacts
with oxygen to form peroxides that degrade the cyt b.sub.6f
activity over time as suggested by Fuerst et. al. (E. P. Fuerst and
M. A. Norman, Interaction of herbicides with photosynthetic
electron transport. Weed Science 39, 458-464 (1991)).
Photoactivity of Mediators:
[0106] Constant potential measurements on unmodified MWNT
electrodes in the presence of mediators at 0.2 V under light on-off
conditions showed that the ferricyanide [Fe(CN).sub.6].sup.3-/4-
redox couple exhibited less photo-response than the benzoquinone
complexes used by others in the literature. Therefore the observed
photocurrent activity in our thylakoid-MWNT composite electrodes
can be directly attributed to thylakoids and not the
[Fe(CN).sub.6].sup.3-/4- mediator.
Absorption Spectra of Mediators and Inhibitors:
[0107] The peak at 673 nm indicates absorbance via chlorophyll-a.
The absorption spectrum for the thylakoid-free MWNT electrode did
not contain the chlorophyll peak (FIG. 12A), indicating that the
mediator does not compete chlorophyll for absorbing light in the
660-675 nm range. Also the thylakoid-MWNT composite electrode in
the presence of mediator showed the 664 nm chlorophyll-a peak,
indicating that the light absorbance of chlorophyll was not
affected by the presence of mediator. Similar absorption spectra
were also obtained for the composite electrodes in the presence of
herbicide, DCMU and the inhibitors, KCN and Paraquat/diquat.
Effect of Light Intensity on Photocurrent:
[0108] The photo-electrochemical response of the thylakoid-MWNT
composite modified electrodes varied with light intensity as shown
in FIG. 13. Initially the system was in dark. A high intensity
light (80 mW cm.sup.-2) was illuminated after 60 sec, which
resulted in the increase in measured photocurrent at 0.2 V to a
stable value of 2.6 .mu.A. At time 540 s, the light intensity was
decreased to "medium" setting in the lamp that resulted in the
decrease in photocurrent to 1.9 .mu.A. When light intensity was
further decreased to "low" setting, the photocurrent of the
composite further decreased to 0.9 .mu.A. The results demonstrate
very good dependency of thylakoid photo-response on the intensity
of the incident light. In these experiments the light passes
through the glass cell and buffer solution to reach the electrode,
not all the intensity of light was fallen on the electrode surface
from the light source.
Advantages of Thylakoid Immobilization:
[0109] To understand if there are advantages associated with
immobilizing thylakoids for carrying out photo-electrochemical
reactions, rather than suspending them in the solution, separate
experiments were performed. FIG. 15A reveals that the immobilized
thylakoids exhibited a fairly stable and reproducible
photo-electrochemical activity over several duty cycles, whereas
the thylakoids suspended in solution showed a gradual loss in the
photocurrent activity with less reproducibility. Moreover, despite
a high concentration of chlorophyll in solution (up to 400 times
more), the photocurrents of suspended thylakoids were significantly
lower than that of immobilized thylakoids. The electron flux of
mediators for immobilized thylakoids would be higher than for
suspended thylakoids due to the proximity of thylakoid membrane
proteins to the electrode, which reduces the diffusion distance for
mediators. It can also be noticed that although high concentration
of suspended thylakoids increases the photocurrents, the trend is
reversed at exceedingly high thylakoid concentrations due to the
issues of high turbidity and low light penetration in the
electrolyte, a case that was carefully avoided in the above
experiments. The cyclic voltammograms (FIG. 15B) showed redox
activities for immobilized thylakoids arising from the direct
interaction of surface bound proteins with the electrode. For the
case of thylakoids suspended in the solution there was no such
redox activity. Therefore some sort of immobilization appears to
enhance electron transfer and high photocurrents.
Steady State Analysis:
[0110] When light was illuminated a large increase in anodic
current was observed due to ferrocyanide oxidation at the electrode
surface. This would require a continuous ferricyanide reduction by
thylakoid membrane proteins in the presence of light. Over time the
current generation stabilized to a constant value at approximately
0.675 pA (FIG. 14A). This indicates that the observed decrease in
photocurrent over time during different light on-off cycles was due
to transience in the mediator diffusion, which reaches steady
state. The observed phenomenon could partly be due to the
establishment of steady diffusion gradients in the system.
Initially there was a high concentration of mediator present at the
electrode-solution interface. Upon illumination, the redox couple
undergoes transition from ferricyanide to ferrocyanide and which
results in a decreased concentration of the ferricyanide at the
interface. This slows down electron transfer to the electrode until
it reaches equilibrium upon which a steady photocurrent was
observed. In the absence of light (FIG. 14B), the photocurrent was
stabilized to .about.1 nA. As we can see from the figures, over
time the currents from both experiments reached the same steady
value. This suggests that the decrease in the photocurrent over
time was partly due to the transience in mediator diffusion.
However a loss of photo-electrochemical activity or composite
dissolution from the electrode surface over time can neither be
verified nor confirmed based on these experimental results.
Herbicide Inhibition of Photosystem II:
[0111] Upon exposing the thylakoid to DCMU herbicide that inhibits
PSII activity, no photo-electrochemical activity was noticed in the
light on-off tests. However, the cyclic voltammograms under light
showed no loss in the redox activities of both cyt-b.sub.6f and
plastocyanin (FIGS. 16A-16B), indicating the direct electrochemical
activity of the thylakoid membrane redox proteins were not affected
by the PSII inhibition.
Non-Involvement of PSI in Photocurrent Generation
[0112] The non-involvement of PSI in photocurrent generation was
studied by inhibiting PSI activity by paraquat/diquat solution
mixture (of the bipyridillum family). The solution mixture was
added to thylakoids prior to immobilization. Paraquat
(E.sub.0=-0.45 V) acts as a competitor to ferredoxin (FD,
E.sub.0=-0.51 V) for accepting the electrons from the
F.sub.a/F.sub.b site of PSI (E.sub.o=-0.56 V) in the photosynthetic
pathway. As shown in FIG. 17, the presence of the PSI inhibitor did
not significantly reduce the photo-electrochemical response of the
thylakoid-MWNT composites. Therefore a portion of the electron flux
generated at the OEC site must have been diverted towards the
electrode through [Fe(CN).sub.6].sup.3-/4- mediator as depicted in
FIG. 1, rather than to the PSI complex via the natural pathway.
CONCLUSION
[0113] The present example demonstrated high photo-electrochemical
activity of immobilized thylakoid-MWNT composites for light energy
harvesting application. The findings have significant implications
for photosynthetic energy conversion and photo fuel production. The
composites exhibited direct electron transfer activity, which can
be enhanced by using a suitable mediator. Control experiments
confirmed that the light-induced water splitting reaction at the
PSII complex was the primary source of electrons for photo-current
generation. At least some advantages of using thylakoid membranes
as opposed isolated photosystems lie in the self-assembly and
utilization of direct electrochemical redox activities of more than
one membrane proteins present in the thylakoid. The thylakoid-MWNT
composite electrode yielded a maximum current density of 68 .mu.A
cm.sup.-2 and a steady state current density of 38 .mu.A cm.sup.-2,
which are two orders of magnitude higher than the previously
reported values in other systems (Table 1). The
photo-electrochemical cell delivered a maximum power output of 5.3
.mu.W cm.sup.-2. No optimization efforts to enhance the power
density were attempted in this work. Accordingly, improvements in
power densities can be realized by engineering optimization such
as, but not limited to, designing suitable membrane-less
electrochemical cells, selecting materials for electrode
substrates, developing superior immobilization methods etc.
Additional understanding of the electron transport pathways will
help enhance direct electron transfer and development of a
mediator-free system to demonstrate direct light to electricity
conversion. The bio-inspired photosynthetic energy conversion
technology using plant thylakoids demonstrated in this example
offers great potential for green energy harvesting based on a
natural process that evolved over millions of years.
References, each of which is incorporated herein by reference.
[0114] 1 R. E. Blankenship, Molecular Mechanisms of Photosynthesis,
Blackwell Science, Oxford, U.K., 2002. [0115] 2 R. E. Blankenship,
D. M. Tiede, J. Barber, G. W. Brudvig, G. Fleming, M. Ghirardi, M.
R. Gunner, W. Junge, D. M. Kramer, A. Melis, T. A. Moore, C. C.
Moser, D. G. Nocera, A. J. Nozik, D. R. Ort, W. W. Parson, R. C.
Prince and R. T. Sayre, Science, 2001, 332, 805-809. [0116] 3 D.
Gust, T. A. Moore and A. L. Moore, Acc. Chem. Res., 2001, 34,
40-48. [0117] 4 McConnell, G. Li and G. W. Brudvig, Chem. &
Biol., 2010, 17, 434-447. [0118] 5 I. Listorti, J. Durrant and J.
Barber, Nat. Mat., 2009, 8, 929-U922. [0119] 6 N. S. Lewis,
American Scientist, 1995, 83, 534-541. [0120] 7 L. Sun, L.
Hammarstrom, B. Akermark and S. Styring, Chem. Soc. Rev., 2001, 30.
[0121] 8 T. J. Meyer, Acc. Chem. Res., 1989, 22, 163-170. [0122] 9
I. J. Iwuchukwu, M. Vaughn, N. Myers, H. O'Neill, P. Frymier and B.
D. Bruce, Nat Nano, 2010, 5, 73-79. [0123] 10 I. Esper, A. Badura
and M. Rogner, Trends in plant science, 2006, 11, 543-549. [0124]
11 K. B. Lam, E. F. Irwin, K. E. Healy and L. Lin, Sens. Act. B:
Chem., 2006, 117, 480-487. [0125] 12 A. Ptak, A. Dudkowiak and D.
Frgckowiak, J. Photochem. and Photobiol. A: Chem., 1998, 115,
63-68. [0126] 13 M. R. Wasielewski, Chem. Rev., 1992, 92, 435-461.
[0127] 14 M. R. Wasielewski, Acc. of Chem. Res., 2009, 42,
1910-1921. [0128] 15 I. Rybtchinski, L. E. Sinks and M. R.
Wasielewski, J. Am. Chem. Soc., 2004, 126, 12268-12269. [0129] 16
A. J. Bard and M. A. Fox, Acc. of Chem. Res., 1995, 28, 141-145.
[0130] 17 R. Bhardwaj, R. L. Pan and E. L. Gross, in Photosynthesis
VI. Photosynthesis and Productivity, Photosynthesis and
Environment, ed. G. Akoyunoglou, Balaban International Science
Services, Philadelphia, 1981, pp. 719-728. [0131] 18 R. L. Pan, R.
Bhardwaj and E. L. Gross, Photochem. and Photobiol., 1982, 35,
655-664. [0132] 19 I. Sanderson, R. Pan and E. Gross, App. Biochem.
Biotech., 1983, 8, 395-405. [0133] 20 K. B. Lam, E. A. Johnson, M.
Chiao and L. Lin, J. Microelectromech. S, 2006, 15, 1243-1250.
[0134] 21 M. Okano, T. lida, H. Shinohara, H. Kobayashi, and T.
Mitamura, Agricultural and Biol. Chem., 1984, 48, 1977-1983. [0135]
22 R. Carpentier and M. Mimeault, Biotech. Lett., 1987, 9, 111-116.
[0136] 23 S. Lemieux and R. Carpentier, J. Photochem. and
Photobiol. B: Biol., 1988, 2, 221-231. [0137] 24 I. Y. Katz, A. Y.
Shkuropatov and V. A. Shuvalov, J. Electroanal. Chem. and
Interfacial Electrochem., 1990, 298, 239-247. [0138] 25 K. Abe, A.
Ishii, M. Hirano and J. F. Rusling, Electroanal., 2005, 17,
2266-2272. [0139] 26 A. Badura, D. Guschin, B. Esper, T. Kothe, S,
Neugebauer, W. Schuhmann and M. Rogner, Electroanal., 2008, 20,
1043-1047. [0140] 27 R. Das, P. J. Kiley, M. Segal, J. Norville, A.
A. Yu, L. Wang, S. A. Trammell, L. E. Reddick, R. Kumar, F.
Stellacci, N. Lebedev, J. Schnur, B. D. Bruce, S. Zhang and M.
Baldo, Nano Lett., 2004, 4, 1079-1083. [0141] 28 I. Katz, A. Y.
Shkuropatov and V. A. Shuvalov, Bioelectrochem. Bioenerg., 1990,
23, 239-247. [0142] 29 S. Tsujimuraa, A. Wadanob, K. Kanoa and T.
Ikedaa, Enzyme and Microbial Tech., 2001, 29, 225-231. [0143] 30 A.
J. McCormick, P. Bombelli, A. M. Scott, A. J. Philips, A. G. Smith,
A. C. Fisher and C. J. Howe, Energy Environ. Sci., 2011, 4,
4699-4709. [0144] 31 M.-H. Ham, J. H. Choi, A. A. Boghossian, E. S.
Jeng, R. A. Graff, D. A. Heller, A. C. Chang, A. Mattis, T. H.
Bayburt, Y. V. Grinkova, A. S. Zeiger, K. J. V. Vliet, E. K.
Hobbie, S. G. Sligar, C. A. Wraight and M. S. Strano, Nat. Chem.,
2010, 2, 929-936. [0145] 32 M. Vittadello, M. Y. Gorbunov, D. T.
Mastrogiovanni, L. S. Wielunski, E. L. Garfunkel, F. Guerrero, D.
Kirilovsky, M. Sugiura, A. W. Rutherford, A. Safari and P. G.
Falkowski ChemSusChem, 2010, 3, 471-475. [0146] 33 J. Barber,
Quarterly Rev. Of Biophy., 2003, 36, 71-89. [0147] 34 N. Lebedev,
S. A. Trammell, A. Spano, E. Lukashev, I. Griva and J. Schnur, J.
Am. Chem. Soc., 2006, 128, 12044-12045. [0148] 35 T. Noji, H.
Suzuki, T. Gotoh, M. Iwai, M. Ikeuchi, T. Tomo and T. Noguchi, J.
Phy. Chem. Lett., 2011, 2, 2448-2452. [0149] 36 I. Ulas and G. W.
Brudvig, J. Am. Chem. Soc., 2011, 133, 13260-13263. [0150] 37 E.
Fuhrmann, S. Gathmann, E. Rupprecht, J. Golecki and D. Schneider,
Plant Physiol., 2009, 149, 735-744. [0151] 38 C. F. Meunier, P. Van
Cutsem, Y.-U. Kwon and B.-L. Su, J. Mat. Chem., 2009, 19,
1535-1542. [0152] 39 R. Carpentier, Photosynthesis Research
Protocols, Humana Press, Totowa, N.J., 2004. [0153] 40 R. P.
Ramasamy, H. R. Luckarift, D. M. Ivnitski, P. B. Atanassov and G.
R. Johnson, Chem. Comm., 2010, 46, 6045-6047. [0154] 41 N. S.
Parimi, Y. Umasankar, P. Atanassov and R. P. Ramasamy, ACS Cat.,
2012, 2, 38-44. [0155] 42 C. Lau, E. R. Adkins, R. P. Ramasamy, H.
R. Luckarift, G. R. Johnson and P. Atanassov, Adv. Energy Mat.,
2012, 2, 162-168. [0156] 43 I. A. Semenova, J. Plant Physiol.,
2002, 159, 613-625. [0157] 44 M. Salin, Physiol. Plant, 1987, 72,
439. [0158] 45 K. B. Lam, Johnson, E. A., Chiao, M., Lin, L., J.
Microelectromechanical Sys., 2006, 15, 1243-1250. [0159] 46 P. N.
Bartlett, Bioelectrochemistry Fundamentals, Experimental Techniques
and Applications, John Wiley & Sons, Ltd, UK, 2008. [0160] 47
M. Richard and J. A. Pedro, Biochem. Bioph. Res. Co., 1975, 63,
1157-1160. [0161] 48 D. G. Sanderson, L. B. Anderson and E. L.
Gross, Biochim. Biophys. Acta, 1986, 852, 269-278. [0162] 49 R.
Ouitrakul and S. Izawa, Biochim. et Biophy. Acta
(BBA)-Bioenergetics, 1973, 305, 105-118. [0163] 50 Y. Abdollahi, A.
H. Abdullah, U. I. Gaya, S. Ahmadzadeh, A. Zakaria, K. Shameli, Z.
Zainal, H. Jahangirian and N. A. Yusof, J. Brazilian Chem. Soc.,
2012, 23, 236-240. [0164] 51 V. Ivanov and S. Lyashkevich, High
Energy Chem., 2011, 45, 210-213. [0165] 52 O. Yehezkeli, R.
Tel-Vered, J. Wasserman, A. Trifonov, D. Michaeli, R. Nechushtai
and I. Willner, Nat. Comm., 2012, 3, 742. [0166] 53 N. Terasaki, M.
Iwai, N. Yamamoto, T. Hiraga, S. Yamada and Y. Inoue, Thin Solid
Films, 2008, 516, 2553-2557. [0167] 54 V. Bhalla and V. Zazubovich,
Anal. Chim. Acta, 2011, 707, 184-190. [0168] 55 A. Badura, B.
Esper, K. Ataka, C. Grunwald, C. Woll, J. Kuhlmann, J. Heberle and
M. Rogner, Photochem. and Photobiol., 2006, 82, 1385-1390. [0169]
56 S. B. Powles, Ann. Rev. of Plant Physiology, 1984, 35, 15-44.
[0170] 57 P. Sarvikas, M. Hakala-Yatkin, S. Donmez and E.
Tyystjarvi, J. Exp. Bot., 2010, 61, 4239-4247. [0171] 58 I. Baroli
and A. Melis, Planta, 1996, 198, 640-646. [0172] 59 L. N. M.
Duysens, Biophys. J., 1972, 12, 858-863. [0173] 60 J. Ahmed, W.
Park and S. Kim, Bull. Korean Chem. Soc., 2009, 30, 2195-2196.
[0174] 61 K. B. Lam, E. A. Johnson, M. Chiao and L. Lin, J.
Microelectromech. Sys., 2006, 15, 1243-1250. [0175] 62 S. A.
Trammell, A. Spano, R. Price and N. Lebedev, Biosens. Bioelect.,
2006, 21, 1023-1028.
Example 2
Photosynthetic Energy Conversion using Photosynthetic Bacteria
Composites
[0176] In the present example, electrochemical cells were designed
and tested similar to those described in Example 1, above, except
in place of thylakoids, photosynthetic bacterial organism were
complexed with carbon nanotubes to provide photosynthetic energy
conversion.
Materials and Methods:
[0177] Materials and methods are similar to those described in
Example 1 above, except as noted below. Sterile cultures of Nostoc
sp. and AV were obtained from Bioconversion Research Centre, UGA
and cultured in our laboratory in shake flasks using BG11 medium
under 12 hr light/dark cycles. Once the optical density at 750 nm
(OD.sub.750) reaches around 1 (happens .about.15 after the culture
inoculation), the culture was harvested by centrifuging at 5000 rpm
for 10 min at room temperature and washed in phosphate buffer (0.1
M, pH 7) before used for immobilization onto the electrode. 5 .mu.l
of multi-walled carbon nanotubes suspension (1 mg/ml in DMF) was
dropped on the carbon paper and allowed to dry. 5 .mu.l of the
washed bacterial cells were immobilized on the top of carbon
nanotube layer, allowed to air dry. The resulting bacterial cell
modified carbon nanotube electrode was then used for the
photo-electrochemical experiments.
Results and Discussion:
[0178] The morphology of the immobilized Nostoc sp. and Anabaena
variabilis (AV) on the multi-walled carbon nanotubes (MWNT) were
studied by scanning electron microscopy (SEM). FIGS. 18A-18D shows
the SEM images of carbon paper, MWNT on carbon paper, Nostoc sp. on
MWNT, and AV on MWNT. FIG. 18B shows well dispersed MWNT uniformly
deposited on the carbon paper surface forming a mesh like matrix.
The filaments of the Nostoc sp. and AV can be seen in FIGS. 18C and
19D. However, careful analysis of the images reveals that the
sheath wrapping the photosynthetic bacteria has good interaction
with the MWNT (edges touching the MWNT in FIG. 18C). In Nostoc sp.
the interaction of sheath with the MWNT was much higher than at AV.
This interaction of Nostoc sp. with the MWNT could possibly
facilitate higher electron transfer from the electron transport
pathway to the MWNT compared to AV. Electrochemical experiments
were given in the following to verify this hypothesis.
[0179] In general, the value of open circuit potential (OCP) is
dictated by the mixed potential caused by a variety of electron
transfer reactions whose individual redox potentials range from
-1.3 to 1.0 V vs. SHE. FIG. 19A compares the open circuit
potentials (OCP) of Nostoc sp., AV and in the absence of bacteria
(i.e., MWNT electrode). During the course of the experiments, dark
and light conditions (light on-off cycle) were varied alternatively
with an interval of 300 s. During the light on-off cycle, the
potential of Nostoc sp. and AV modified electrodes showed stepwise
variation indicating photo-electrochemical activity by these
organisms. The potential variation between light on-off cycles was
as high as 100 mV for Nostoc sp. and 30 mV for AV. In the absence
of bacteria the electrode showed an OCP of about 0.25 V, and the
OCP was constant throughout the light on-off cycle indicating that
there was no photo-electrochemical activity on the bare MWNT
modified carbon paper (CP) electrode.
[0180] The photo-activity of the photosynthetic bacteria modified
electrodes were evaluated at constant working electrode potential
of 0.2 V and the corresponding photo-current response was measured
over time during light on-off cycles. FIG. 19B shows the steady
state photocurrent density obtained from the photosynthetic
activity of both Nostoc sp. and AV. This increase in current
density during light-on clearly indicates the transfer of electrons
from the photosynthetic bacteria to the MWNT and eventually to the
electrode surface. The natural electron transport pathway of the
linear photosynthetic process starts with water oxidation at the
oxygen evolving complex (OEC) site of the PSII complex. The
electrons are then transferred from here to the PSI complex through
plastoquinone, cytochrome (Cyt)b.sub.6f and plastocyanin
respectively. In this pathway, before the electron reaches PSI, any
redox protein except PSII can transfer electrons to the MWNT, and
thus, to the electrode. The maximum photocurrent density was 35 mA
m.sup.-2 for the Nostoc sp. modified electrode, and 10 mA m.sup.-2
for the AV modified electrode.
[0181] The current vs. time plots of fixed anode potentials were
used as a tool for optimizing the photosynthetic bacteria loading
on the electrode surface. The results showed that the photocurrent
density was directly proportional to Nostoc sp. loading (FIG. 20).
The maximum loading achieved on the CP-MWCNT electrode during this
test was 56 .mu.g. However, higher loadings could be achieved by
modifying the morphology and physical properties of the MWCNT
coating on the CP electrode.
[0182] In the case of AV (FIG. 21), a maximum loading of 128 .mu.g
was achieved. Comparison of Nostoc sp. and AV results reveals that
Nostoc sp. Appears to possess higher photo-electrochemical activity
than AV under the conditions tested. Since Nostoc sp. performed
better among the two organisms studied, it was selected for
detailed characterization studies.
[0183] The photo-electrochemical response of the Nostoc sp. varied
with incident light intensity as shown in FIG. 22. Higher
illumination resulted in higher photocurrent density, in the order
of 27<50<76 mW cm.sup.-2. The results demonstrate the
dependency of Nostoc sp. photo-response on the intensity of the
incident light. It should be noted that in the experimental set up
for this example, only a small fraction of the incident light
actually fell onto the electrode. This is because the electrode was
kept in a hanging position inside a glass electrochemical cell
containing the buffer solution and there was no attempt made to
funnel the light to the electrode surface.
[0184] The stability of current generation was studied by observing
the steady state current change during the experiments. To identify
the steady state, currents were measured in continuous light and
dark conditions without cycling. FIG. 23 shows the overlay of light
on-off cycle results with that of steady state studies at
continuous light and continuous dark conditions. As seen in FIG.
23, over time, the currents from both experiments reached the same
steady value. The steady state photocurrent density for Nostoc sp.
was 10 mA m.sup.-2.
[0185] The major pigment present in all known photosynthetic
organisms is chlorophyll a, which forms the reaction centers in
both PSII (P680) and PSI (P700), absorbing light efficiently at 465
nm and 665 nm. Additionally, cyanobacteria such as AV and Nostoc
also possess certain accessory pigments such as phycocyanin,
phycoerythrin and carotenoids that maximize the range of action
spectrum by absorbing a range of wavelengths other than that
absorbed by Chlorophyll a (Chl a). Upon absorbing the
characteristic light, these accessory pigments transfer the
absorbed energy to other pigments and finally to the reaction
center Chl a. FIG. 24A shows the action spectrum in the visible
region for Nostoc sp. and AV, containing distinctive peaks
corresponding to absorbance for Chl a, phycocyanin, and
phycoerythrin. Various insights can be achieved by analyzing the
generation of photocurrent in Nostoc sp. by illuminating with
lights of different characteristic wavelengths, so that the
contribution of different pigments towards photocurrent generation
can be studied. Experiments have been conducted using lights of
440, 500, 550, 600, 640 and 680 nm wavelengths as shown in FIG.
24B. It has been found that Nostoc sp. illuminated using 640 nm
light (corresponding to phycocyanin) resulted in maximum
photocurrent.
[0186] The mechanism of electron transfer from the photosynthetic
electron transfer chain (PETC) to the MWCNT was also studied with
the help of photosystem inhibitors. Inhibitors such as DCMU
((3-(3,4-dichlorophenyl)-1,1-dimethylurea), DBMIB
(2,5-dibromo-3-methyl-6-isopropylbenzoquinone), KCN (potassium
cyanide) and antimycin-A specifically block a particular site of
the electron transfer chain (FIG. 25). DCMU binds at Q.sub.B site
of PSII and inhibit the electron transfer downstream of PSII. DBMIB
is an analogue of PQ (plastoquinone) and binds at the Q.sub.0 site
of cytochrome b.sub.6f complex, arresting the electron flow beyond
that complex. KCN has been found to replace the copper ion of
plastocyanin (PC), thereby preventing the electron flow from Cyt
b.sub.6f to PSI. Antimycin-A blocks the electron transfer from
ferredoxin to PQ, disrupting the cyclic electron transfer around
PSI. All these inhibitors are highly site-specific and have been
used in the photosynthesis research to decipher the source of
electron channeling from the photosynthetic machinery.
[0187] Experiments were conducted to measure the photocurrent
produced by the Nostoc sp. after incubating the cells with the
inhibitors such as DCMU, DBMIB and KCN at varying concentrations,
and the results have been summarized in FIG. 26. DCMU was found to
inhibit the photocurrent by around 80% compared to that of wild
type as shown in FIG. 27, indicating that the primary source of
photocurrent is the electron from oxidation of water by PSII. The
remaining 20% photocurrent could be attributed to either lack of
complete inhibition by DCMU at the Q.sub.B site of PSII, or the
electron could have come from possible sources other than PSII.
[0188] The inhibition by DBMIB is highly dependent on the
concentration of the DBMIB used as illustrated in FIG. 28. At a
concentration of 0.1 mM, DBMIB inhibits the photocurrent
completely, whereas upon increasing the concentration to 1 mM, the
generation of photocurrent is enhanced significantly compared to
that without inhibitor. It has also been observed that a low
concentration of 0.01 mM DBMIB is not sufficient enough to arrest
the electron flow at Cyt b.sub.6f complex. Due to the complete
reduction in photocurrent at 0.1 mM DBMIB, it is believed that the
Cyt b.sub.6f is the site in PETC through which the electrons reach
the electrode via MWCNT generating photocurrent.
[0189] Inhibition of photocurrent by KCN is considerable (FIG. 29)
with nearly 50% reduction in photocurrent compared to the wild
type. If the major detour of electron towards the electrode is
through Cyt b.sub.6f, the inhibitors downstream of it should not
diminish the photocurrent. However, it is not so in the case of
KCN, which indicates that, the total photocurrent has not
exclusively been arising from the electron leaving Cyt b.sub.6f
site. Rather, an alternate pathway could be involved in
contributing to the photocurrent. The cyclic electron transport
(CET) around PSI can be one other contributing factor for the total
photocurrent. Experiments with chemicals inhibiting the CET such as
antimycin A will be helpful to analyze contribution of cyclic
electron transport to the generation of photocurrent.
[0190] The Q cycle catalyzed by Q.sub.0 and Q.sub.i sites of Cyt
b.sub.6f complex represent another possible source for the
photocurrent. The two electrons coming from the oxidation of
PQH.sub.2 at Q.sub.0 site of Cyt b.sub.6f complex are not
completely transferred to PC; rather only one electron is
transferred through Cyt f to PC, and the other electron is used to
reduce PQ at Q.sub.i site through atypical heme x (Zhang et al,
2004; Stroebel et al, 2003). It has been investigated that this
heme x near the Q.sub.i site functions as a redox wire allowing
ferredoxin or other electron carrier to reduce PQ pool.
[0191] The foregoing examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is in
atmospheres. Standard temperature and pressure are defined as
25.degree. C. and 1 atmosphere.
[0192] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. In an embodiment, the term "about" can include
traditional rounding according to measurement techniques and the
numerical value. In addition, the phrase "about `x` to `y`"
includes "about `x` to about `y`".
[0193] Many variations and modifications may be made to the
embodiments described in the preceding Examples. All such
modifications and variations are intended to be included herein
within the scope of this disclosure and protected by the following
claims.
* * * * *