CA1099332A - Solar powered biological electric cell using mesophyll cells - Google Patents

Abstract

SOLAR POWERED BIOLOGICAL ELECTRIC
CELL USING MESOPHYLL CELLS

ABSTRACT OF THE DISCLOSURE
A solar powered biological fuel cell is provided which is capable of generating a direct electric current in response to incident solar energy. The cell includes a suspension of mesophyll cells isolated from a C4, malate forming species such as Digitaria sanguinalis (crab grass) or mesophyll cells isolated from what are termed Crassulacean Acid metabolism (CAM) species, malic enzyme, a nicotinamide adenine dinucleo-tide, a xanthine oxidase enzyme, a potential mediator such as methylene blue; and a catalyst such as pyruvate in an aqueous solution; and an appropriate electrode assembly.

Description

~Q9~32 SOLAR POWERED ~IOLOGICAL RLECTRIC
CELL USING MESOPHYLL CELLS

BACKGROUND OF THE INVENTION

I A fuel cell is an electric cell that converts the chemi-cal energy of a fuel directly into electric energy in a continuous~
process. The efficiency of this conversion can be ma~e muah greater than that obtainable by thermal-power conversion. In the latter the chemucal reaction is made to produce heat by combustion. The ~ heat is then transformed partially into mechanical enerby by a heat engine, which drives a generator to produce direct current.

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33~ -, `1 ¦ Although/ in principle, the natur~ of the reactants is ,limited, the fuel-cell reaction usually involves the 3 j combination of hydrogen with oxygen, as shown by Equation (1).

4 ¦ At 25C an~ 1 atmosphere pressure, that is, standard temperature and pressure (STP), the reaction takes place with a free energy 6 change (~ G) of ~ G = 056.69 kcal/mole, that is, ~37,000 joules/

7 mole water. . ~. . -8 .. . -~ . . . .
9 H2 tg) ~ 1/202 (g~ 7' H2 (/) , , , (1) tl - - ~ -. ' ~
12 If the reaction is harnessed in a galvanic cell working ~3 at 100% efficiency, a cell voltage of 1.23 ~olts results~ Xn ~4 actual service such cells have shown steady-state potentials in thc L5 range 0.9-1.1 volts, with repor~d coulombi~ efficiencies of the 17 oxder 73-90%.
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~9 Fuel cells are of 200-500 watts capacity and 50-100 '.?.0 ma/cmZ current density. Larger prototypes have been produced, ~1 some as large as 15 kw capacity.
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'4 ~ The most successful prior art type is the H2-O2~fuel 3~ cell of the direct or indirect ~ype~ In the direct type, hydrogen `6 and oxygen are used as such, the fuel being produced in independent .~ installations The indirect type, employes a hydrogen-yenerating .~ unit which can use as raw materi21 a wide variety of fuel~ The reaction taking place at the anode is as in ~:q. (2), and at the cathode as in Eg. (3/. . - , ;~ . . ' .
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1 I 2H2 ~ 40X~ 4H20 + 4e- (2)

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4 ~ 2 + 2H20 ~ 4e ~ 40H- ~ (3) sl . - , .,.~- . I
6 ¦ Because of the low solubility of H2 and 2 in electro-q ¦ lytes, tlle reactions take-place at the interface electrode-B electrolyte, requiring a lar~e area of contact for a large elec-9¦ tron flow. This is obtained with porous materials called upon to ¦
~C¦ ful~ill the following main duties: The ma.erials must provide 11¦ contact between electrolyte and gas over a large area, catalyze ~21 the~reaction, maintain the electrolyte in a very thin layer on 13 ¦ the surface of the electrode, and act as leads for t~e trans-~4l mission of electrons. .
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6¦ Sun powered photosynthetically-driven biologlcal fuel ~¦ cells are also known to the prior art. In U. S. Patent 3,~77,879,`

8¦ a device is described in which an electrical fuel cell is formed 191 by using two chambers, one placed in sunlight and supplied with 20¦ nutrients and microorganisms ~hich trans~er light energy o~ photons!

21¦ into chemical ener~y in the form of algae or carbohydrate, and the 22¦ other placed in the dark where the chemical energy is release~ by ~31 reducing bacteria which produces compounds which release electrons.

~4 A bridge is included in the device to provide a pathway for cationsl 26 and anions without a transfer of material he~ween chambers. Elec- ¦

27 trons are released to an anode of the device by sulfites ~nerate~¦

28 from sulfates by bacterial action~ The energy of this action is derived from the sun and stored as bacterial metabolites, these 2~ bei~g ~ed .o the bacteria to drive the reduction reactions generat-! ing compounds which, in turn, give up electrons to an electrode 31¦ element.
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~ The optimal condition for a photosynthetically-driven æ ~el cell would be one i~ which the cells collecting sunlight h~d 3 as their genetic-based biochemical directive that most of the

4 photonic energy captured within the chloroplasts of the cells 6 (assuming eucaryotic cells are used rather than photosynthetic 6 bacteria or blue-green algae) would be exported from within the 7 living cells to outside of ,the cell organism, where it could be 8 acted upon without further catabolism by any other organism to produce electrons with a negatlve standard reduction potential as 0 close as possible to hydrogen ~-.42 volts).
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13 '- Work with chloroplast preparations has shown that it is 14 possible to produce molecular hydrogen with certain enzymes in the absence of appreciable oxygen tensions from ~unlight. , 16 '- , " , ,, ` ' '' ', - ' ' , ' `"
17 ' ' '; '- - ` ` ' ` ' , 18 Qxygen (+.82 volts) produced by the water-splitting 1~ activity of photosynthesis would constitute a readily-available source of oxidant, and should be thought of as the oxidant of choiqe ~1 for accepting electrons at the cathode, whether the cathode is 22 separated from the living,cells to which oxyyen is deli~ered, 23 or is spatially set among the cells to which oxygen diffuses.
~4 ' "' " ' ,' ~5 , ~' ' ' ' ' ' ' ' 26 In photosy~thesis, four photons captured by a chloro-~7 phyll pigment system with an averaye energy of approximately 28 50 Xcals per einstein are needed to reduce one molecule of nico-~9 tinamide adenine dinucleotide phosphate (NADPH) at approximately 53 Kcals per mole. Therefore, the theoretical maximum conversion 3~
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~ 33 lli or photonic ener~y to reducing potential is ap~roximately 25%.

2 Tapping the energy as formed into carbohydrate leads to another 3 reduction in the theoretical efficiencyO The actual efficiency of photosynthesis in nature for recovered energy in fixed carbon for a field of sugar cane, one of the most efficient species, is 6 as high as eight percent. - . . ; .
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. ~ A cell type.which approaches ~hese model characteristics 0 was first described in the pxior art as a type of cell living within the leaves of certaln highly photosynthetically-efficient ~2 tropical plants s~ch as crab grass or sugar cane. In the field 13 of plant physiology, what is termed Kranz-type leaf anatomy has 14 been-described in the prior art in which the vascular bundles are ¦
surrounded by two concentric chlorophyllous layers, thereby formin~
16 an inner parenc~yma bundle sheath layer and an outer mesophyll 17 layer. It has also been found that these species wi~h Kranz-type 18 leaf anatomy also fix carbon dioxide into four carbon compounds 19 such as oxaloacetate, aspartate or malate, rather than by Calvin ~0 cycle type C02 condensation with ribulose bisphosphateO These species are described as "C4" type plant species. "C~" plant: :
species are further segregated into what are termed "malate 23 farm~rs" or "aspartate formers n depending upon which co~.pound 24 appears to be the major immLdiate product of carbon fixation.

26 . .
27 It was also noted in the prio~ art that this newly-28 discovered ~Ddè of carbon dioxide formation was associated wit~
2g Kranz-type leaf an~tomy. Further investigation revealed ~hat the 31 .

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l ¦ ~esophyll cell type was responsible for the carbon dio~id~ fi~a- j tlon reaction, and that this cell was thought to transport c~rbon 3 dioxide- and reducing equivalent derived from the sun to the 4 neighboring bundle sheath cell type. The mesophyll cel~ collect~

carbon diQxide and sunlight, while the bundle sheath cells 6 spe_ialize in carbohydrate formation from the carbon dioxide and 7 energy in malate which is tlansported to the bundle sheath cells.

8 Another prior art finding is tha~ mesophyll cells from C4 species have reduced photo-respiration rates which, in other plant species, 0 use Up energy and reduce the overall conversiOn efficiency of the energy in photons to energy in chemical bonds. The C4 plants ~2 also are more efficient users of solar energy at high light inten-13 sities than p~ant species using the first discovered means of 14 carbon dioxide fixation, such as spinach.

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17 Recent investigations in the f.ield of plant physiology 18 have demonstrated that mesophyll cells, from what are termed "C4 9 malate formers" in the scientific l:iterature, have th~ possibility 2 of producing extracellular reducing e~uivalents at the level of 2 malate wh ch can be transported through a series of oxidation and 2 reduction reactions to an inert electrode. A suspension of meso-2 phyll cells isolated from the leaves of these species export malat~

24 and absorb pyruvate and C02. The oxygen (~.82 volts) produced from the water-splitting activity in photosynthesis can be used 26 to accept electrons at a cathode.

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3~ l ~ ithin "C4 n type mesophyl~ ce~ls, the NADPH formed in 2 ~ chlorplast is used to reduce oxaloacetate to malate which is 3 I transported to bundle sheath cells which in nature lie next to 4 mesophyll cells in the leaf. By eliminating the bundle sheath cells and using malic enzyme, malate c~n be oxidatively decar-6 boxylated to pyru~ate which is then shuttled back to the mesophyll 7 ! cell and serves as a precursor of phosphoenol pyruvate production 8 I and, hence, malate formation within the mesophyll cell. Thus, we¦
are ~ble to interdict the normal flow or reducing equivalents at the NADP~ (-.32 volts) level and use ~he energy to produce a cur-l 11 rent of electrons. By the use of malic enzymes (L-malate: NAD
12 or NADP oxidoreductase (decarboxylating) EC 1.1.1.38 or 13 EC 1.1.1.40), the extracellular malate can be converted to C02 14 and pyruvate as two electrons are transferred to reduce a nico-tinamide adenine dinucleotide INAD(~.
6 I " -17 ~ -181 TAe technology or transferring ~lectrons from extra-19 cellular NA~PH to the electrode element of a fuel cell has been demonstrated in the prior art, UsiIlg a potential mediator sub-21 stance tbenzyl viologen) to transfer electrons from NADPH to an 22 electrode to measure the standard reduction potential of the 23 ~DPH~NADP~ couple. It was found that xanthine oxidase was neces-~4 sa~y to catalyze the reaction. Other workers in the art have used other flavoprotein NADH and N~DPH dehydrogenases to catalyze 26 the transfer of electrons from t~ese two compounds (NADH ænd 77 NADPH) through various reducible dye intermediates and other re-28 ducible compounds~ such as quinone, which serve as potential media tor compounds for delivery of electrons to an inert electrodeO
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~¦¦ Methyl phenzzonium methosulphate and similar 2 ! compounds have also been used to transfer electrons from the nico-3 tinamide adenine dinucleotide and the phosphate analog, NADPH, 4 directly to a ruel cell electrode without the necessity of an intervening enzyme step. Malate coming from C4 mesophyll cells 6 in some wayl must be made to g~ve up electrons and form pyruvic 7 acid (as in the case of m~late, pyruvic acid is the acid form of 8 pyruvate; the form is dependent upon the hydrogen ion concentra-tion of the surrounding environment), which is reabsorbed by mesophyll cells and serves as a precursor to malate formation 11 within the mesophyll cell. Malic enzy~es (L-malate: NAD oxido-12 reductase (decarboxylatingl EC 1.1.1~38 and EC 1.1.1~40) are kno~
13 to exist in multiple ~orms. It can be isolated from bundle sheat~
14 cells from C4 type plants, lactobacillus bacteria, cactus, or other species. Malic enzyme catalyzes the following reversible 17 reactions:
Mn~+ or k~g++
18 L-Malate + NA~P¦ C~ + Pyruvate + C02 + NAD (P ~ H

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21 The equili~rium constant fa~ors malate formation when 22¦ C~2 is at a pressure of 760 mm of Hg and is 5xlO 2mole 1. Pyru-23 vate and C~2 are taken up by mesophyll cells, and NAD~ or NADP+
24 is reformed as ~ADH or NADPH gives up electrons which ~ind their 25 ¦ way to an electrode; thus, the reaction can be m~de to go toward 26 ¦ malate oxidation as the products of the reaction are removed as 27 current flows through the circuit. ~s previously mentioned 28 there are several methods to transfer electrons from the nico-29 I tinamide adenine dinucleotides to the fuel cell electrode. An 30 ` i~portant consideration in the transpGrt ~f the two electrons ~ ~ .

l `` ~ 33~ 1 1 ¦ fJ `' NADH or NADPH to an electxode assem~ly is that the xanthine 2 oxidase catalyze step as well as the donation of electrons step 3 at the surface of the electron accepting electrode assembly must 4 be conducted in the absence of high oxygen tension in the aqueousj solution because oxygen can serve as the electron acceptor at both 6 1 of these reaction surfaces. Methyl phenazonium methosulphate is 711 also susceptible to oxidation by molecular oxygen.
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Tissue culture technology has progressed so that the ~1 means of growing and maintaining cultures of cells of the meso- i t2 ¦ phyll cell types has evolved. When urnished with the right con-j 13 ¦ centrations of inorganic salts and organic growth-stimulating 14 I substances, mesophyll cells have been kept autotrophic for exten-15 ¦ ded periods as described in the prior art.

17 I - ` -18 Enzymes used in the biological fuel cells under consi-19 deration must ~e kept from proteolytic attack by bacterial agents contaminating the suspension of living mesophyll cells for an 21 extended function o the device. This may ~e achieved by using 22 polyacrylamide cross-1inked polymers or other po1ymeric compounds 23 capable of forming a clathrate type molecular cage around the pro-24 tein enzyme, Co~alent linkage o~ the malic enzyme to the lattice structure may or may not be necessary depending upon compounds and 26 techniques employed. If a second enzyme is employed to transfer 27 electrons from the reduced nicotinamide adenine dinucleo~ide to 28 a potential mediator substance, this protein also should be en-29 closed within a protectiYe element.
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oqs~32 1 i The mesophyll cells can be isolated from what are terme 2 C4malate-formin~ species of plants by either enzymatic methods, 3 .as described by Jense, Plant Physiol. 48 9-13, 1971, or Gnanam 4 and Ku~andaivelu, Plant Physiology 44: 1451-1456, 1969, and adapted toward separating C~ mesophyll cells by various plant 6 physiologists or grinding techniques as employed by Edwards and . Black, Plant Physiology 47: 149-156, 1971, can be used to free 8 mesophyll cells from other cells of the leaf such as bundle sheath ~9 cells. Sin~le mesophyll cells of ~igitaria sanguinalis are appro ximately 15-25 u meters in diameter and can be separated from the `11 ¦ other cell types by passage of solution containing the cells 12 ¦ through nylon filtration net, of approx. 30 u meters pore size, I
13 ¦ which passes mesophyll cel~s but not bundle sheath cells and bundle 14 ¦ sheath strands of cells. Mesophyll cells can be separated from chloroplasts and broken cell.fragments and ~ost bacteria by catch 16 ing the cells on a 10 u meters net~ Further purification of meso'l 17 phyll cell cultures and the development of a cultule from a single 18 mesophyll cell used to cione the mesophyll cells used for the in-l 19 .~ention is within the technology. Best resuts are achieved when 21 cells are ~solated from the leaves of plants with seeds surface ~ sterilized in 1.8% NaClO4 (sodium hypochlorite, bleach) for 10-15 22 minutes and gexminated and grown on sterile ayar containing essen 23 tials salts such as "~OGALANDS"( ) salts, or commercial hy~ro-24 ponic garden salts. . . , . ;.
26 ~e present invention is concerned with an improved type 27 of photosynthetically-driven biological fuel cell. The invention 2B relates specifically to a biological fuel cell for tran~ducing the energy in sunliaht auanta into a useable direct electric cur-~¦ rent. This is achieved by the action of sunlight upon living 31 ¦ cells whose geneti~ makeup and differentiation dictate that the ~ cell~ will export to the extracellular space a large fraction of ..
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the reducing equivalents generated by sunlight in -the chloro-plasts of the cells. The fuel cell of the invention uses photo-synthetic cells isolated from the leaves of Digi-taria sanguin-alis (crab grass); however, mesophyll cells from perhaps many photosynthetic plant species may be used in which malate (malic acid) is produced and appears in the extracellular space.
Th~ls broadly, the inventioll conte~ la~cs a pl~otv-synthetically-driven biological fuel cell which compxises means forming a growth chamber having a transparent -top exposed to sunlight, with a nutrient aqueous solution contained in and growth chamber, and an anode electrode and a cathode electrode mounted in the growth chamber immersed in the sol~ltion in isolated relationship with one another and electrically con-nected to an external load. A plurality of isolated mesophyll cells are suspended in the nutrient aqueous solution, wi-th the mesophyll cells producing malate when illuminated by sunlight, and with nutrlent solution being capable oE sustaining the isolated mesophyll cells in an autotropic state and oE
initiating malate export from the illuminated mesophyll cells into the solution. ~ selected substance is s~sp~ilded in tlle nutrient solution to act on the malate to form NRD~I, and a potential mediator substance is suspended in the nutrient solution to transfer electrons from the NADII to the anode.

BRIEF DESCRIPTION OF THE DRAWING
.
FIGURE 1 is a schematic representation of a biological fuel cell incorporating the concepts of one embodiment of the invention.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT
___ _ The invention consists o a first anaerobic anode electrode 10 and an aerobic cathode electrode 12, each approxi-mately 2" X 2", placed in a container 11 having beell m~dc o~
transparent plastic plates. ~ salt bridge 14 divides the container _ into an upper cathodic chamber 40 which contains the cathode 12 and a lower anaerobic chamber 17 which contains the anode 10. The electrodes 10 and 12 are electrically con-nected to an external load 13. Electrode 10 is formed of platinum metal, and it may be coated with "TEFLO~",( ) or other electrode protective agent.
Mesophyll cells 16 isolated from or grown from the isolated cells of a "C4" type photosynthetic species plant, such as Digitaria sanguinalis, are suspended in a nutrient aqueous solution 15 in a growth chamber 19. Chamber 19 has a transparent top 18 and a bottom 16. A system of catalysts and potential mediators are also suspended ~ -lla-~. ' .
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~ he solution 15 to transfer electrons from malate which is produce~
21 ~ the cells to the electrode 10. The solu~ion 15 is pumped throu~h 3 the chamber 11 by way of pipelines 50, 51 and a pump 7.
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As sunlight impinges upon the mesophyll cells through trans-6 parent top 18. chloroplasts use the light energy to split water (photo 7 system II activaty also know~ as the Hill reaction) thus generating 8 molecular oxygen which is consumed to form OH ions at electrode 12 9 which migrate along a pathway through the salt bridge 14 to the anae-10 robic chamber 17. The growth chamber 19 in which the mesophyll cells 11 ar~ maintained in the living state can be tubular, such as glass pipe,¦
12 or flat covering area, such as a shallow pond with the transparent 13 sunlight transmitting top 18, such as shown in FIGURE 1.
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The nutirient solution 15, within the growtll cha]l~er 19 ma~! ;
16 approximate that of Chandler, Marsac and Kouchkovsky tcan- J. Bot.
17 50: 2265~2270) or Murashige and Skoog Salts (GIBCO) that is, it may 18 be of a type which is capable of sustaining isolated cells in an auto-19 trophic state. Pyruvic acid at approximately 5m molar (5x10 3m) con-20 centration is needed to initate malate export to the extracellular 21 space by the mesophyll cells isolated from C4 photosynthetic, malate 22 forming plant species under illum~nation.
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24 The L-malate (the anion of malic acid) that the cells pro-25 duce is acted upon by malic enzyme 24 within the anaerobic chan~er 17 ¦
2G (L-malate: NAD(P) oxidoreductase (decarboxylating) EC 1.1.1.38 or 27 I EC 1.1.1.40), purified by known means. The enzyme may be incorpor-28 lated into a latice structure of a polymeric compound, such as polycry-29 lamide, or other clathrate producing polymer. The lattice serves to 30 protect the enzyme from enzymatic degradation, loss through surface ~ -31 denaturation, and loss through solubility. The purpose of the re- ¦
32 action of malate and NAD Or NADP iS to yield carbon dioxide, pyru- l 33 vate and a reduced compound, NAD(P)H.

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ll ~s is characteristic of mesophyll cells from "C4" photo-2 synthetic species of plants with Xranz~type leaL anatom~, these 3 cells absorb the pyruvate and carbon dioxide which are products 4 of the malic enzyme catalysized reaction. The export of reducing equivaients being produced from sunlight within the cells is 6 dependent upon pyruvate and CO2 uptake of the cells. Over time 7 malate produc~ion appears stoicimetrically coupled to pyruvate 8 uptake on a one to one basis.
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t l . . The NADH forned by the action of malic enzyme (there 12 are also forms of malic enzyme which react with NADP+~ upon ~alate 13 is used to donate two electrons to a compound termed a potential 14 ! mediator substanceJ such as benzyl ~iologe~ or methylene blue, which in turn give u~ electrons to ~he anode 10 which has a rela-16 tively large surface~area. Most potential mediator compounds are 17 reducable dye compounds such as methylene blue, benzyl viologen 18 or methyl ~iologen. Quinone is also mentioned as a potential 19 mediator substance. NAD~ or NADP+ is thus regenerated, as NADH
or NADPH gives up its two electrons (-.32 volts) to the potential, 21 mediator substance. This potential mediator step can be cata-lyzed by the action of xanthine oxidase with benzyl viologen, or 23 methylene blue. The reaction at xanthine oxidase must be cor.-24 ducted in the absence of oxygen because xanthine oxidase will cause NADH or NADPH to give up electrcns to oxygen directly thus !
26 short circuiting operation of the fuel cel~.

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1 There are several means by which an anaerobic region can be 2 generated in front of anode 10 so that the xanthine oxidase step 3 and the electron donation step to anode 10 can be protected from energy-using oxygen consumption. One method is to place the xanthine oxidase 20 in the anaerobic chamber 17 and to place part of chamber 1.
6 divider 22 over the entrance of the pipeline 50 which ~orms a fluid 7 pathway to electrode 10. The chamber divider 22 is porous to ions 8 and solution molecules; but is not porous to cells. It may be com-9 posed of one or more substances such as a metal and filtration sub-stance.
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12 ', 13 Solution circulation through the pipelines 50, 51 and 14 anaerobic chamber 17 is maintained by pump 7. Rubber gaskets or seals 9 are provided on each side of salt bridge 14. Nylon threaded 16 rod and hex-head nuts 8, hold the electrode and cathodic chamber 17 assembly together.

Another method for generating the anaerobic region in ~1 front of anode 10 is to harvest energy from the ~uel cell at night, 22 or in the dark, so that the mesophyll cells themselves will contri-~3 bute to the generation of the anaerobic region through their own 24 respiration, which consumes oxygen at a rate of about 5% of the rate 25 of their oxygen generating capacity in the light. Most of the oxy-26 gen generated by the cells floats to the surface of the growth cham 27 ber and can be removed from the aqueous interface by pumping.

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l ~ ?332 1¦ A combination of the aforesaid three methods, or other ~¦ methods, can be used to remove dissolved oxygen from contact with 3¦ the anode 10 and xanthine oxidase 20. Two such methods, for example, 41 are to bubble a gas, such as nitrogen to sweep out most of the oxygen¦

5 ¦ and to release a partial vacuum through a couplter 5 and chamber 6.
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8 If an enzyme proves necessary to cataly~e any step, 9 optimally, it should also be insoluablized and protected from de-naturatiOn by incorporation also into a lattice structure. If 11 a potential mediator substance is used that does not need an 12 enzyme catalysis, then this enzyme step may be eliminated. On 13 large volume systems employing mesophyll cells to produce oxidizable 14 malate, the replacement of decaying catalytic activity from en~ymes 15 ¦ needed for electron shuttle steps could prove costly.
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18 ¦ The reduced form of the potential mediator substance 19 ¦ yields electrons to the anode 10. As the reduced potential media-20 ¦ tor gives up electrons to the anode 10, the mediator becomes 21 ¦ o~idized and is then capable of transporting more electrons to 22 the anode.

The cathode 12 can operate on an alternate oxidant, but 26 since oxygen is produced by meophyll cells, and since oxygen is 27 readily available in an earth atmosphere, oxygen is used for an oxi-28 dant Oxygen bubbles formed during illumination of the mesophyll 29j 321 . I ' 'l ~. I

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l ¦cclls float to the surface of the growth chamber 19 due to gravity 2 ¦and are used to furnish oxygen to the surface of cathode 12 at 3 1a partial pressure of approximately one atmo$phere, or air can be 41 used. In systems where the cathode is set away from the cell sus-51 pension it operates optimally in a solution of 32 w/o KOH or NaOH
61 àt 80 C, but will operate ln less basic conditions and cooler tem-8 peratures.

lO¦ The foregoing action produces a potential difference across ll the electrodes 10 and 12 which causes an electrical current to flow 12 through the load 13.
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Malate is stable in solution unless acted upon by enzyme 16 and cofactor complex. Stoichiometrically for each coulomb of elec-17 trons which react with oxygen (2) at the cathode 12, 5 micromoles 18 of electron acceptor is generated in theanaerobic region ne~} ano(le l9 10 tassuming two electrons donate per molecule of potential mediator oxidized.) 23 During operation of the fuel cell of the invention, the 24 l-ate of carbon dioxide fixation by the mesophyll cells sets the theoretical limit of malate formation with the cells, and hence 26 the amount of malate that can he expected to be exported from the 271 cells. During steady state operations the rate of malate export 28¦ has been found to be somewhat less than the rate ~f pyruvate uptake, 291 although the rate of pyruvate uptake appears to be equal to the rate of CO2 fixation. The rate of malate utilization is coupled to 31 pyruvate formation and NADH formation by the malic enzyme step.

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1 NA~H formation from NAD is stoichiometrically coupled on a one-to- ¦
2¦ one basis wi-th malate utilization. Malate will accumulate in the 31 growth cham~er without NAD+ and malic enzyme. The oxidized form of 4 the potential mediator substance is reformed at the anode 10 as the 5 reduced form gives up electrons. As the reduced form of NADII, or

6 the potential mediator substance, gives up electrons, the oxidized

7 form is thus regenerated, thereby ~eing able to shuttle two more 81 electrons in cycle form.

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11 By the use of mesophyll cells, isolated and/or growll from 12 "C4" type photosynthetic plants species, such as Saccharum or 13 Digitaria, a nearly closed system can be achieved, where the net 14 effect is that sunlight is used by the cells to split water during 15 photosynthesis. The electrons taken from water eventually find 16¦ their way back into water after traveling through a circuit of 17¦ approximately 1 volt potential dif~erence between NADH and water 18¦ and doing work. Intermediate compounds in the system participatc 19 ¦as catalysts.
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~21 CAM plant species "CAM" standing for crassulacean acid ~31 metabolism, named for a family of cactus or succulents, may be used ~ to form the mesophyll cells; in which a type of carbon dioxide 25 Ifixation similar to that described for C4 plant species occurs, 26 èxcept that the cycle is internalized within a single cell and 27 operates between the vacuole of the plant cell and the cytoplasm.

2~ Malate is stored in the vacuole. Massi~e (100-200 ueq g 1 fresh ~9 weight) amounts of malate are produced during the dark (from energy 30 captured during the day ) by these cells. Malate can be made to 31 leak from cells by (1~ osmotic shock; (2) use of àgents such as ~Q~332 DMSO (dimethyl sulfoxide) which are known for increasing the permeability of biological membranes; and (3) detergents in low concentrations such as "TRITON"~ )X-100.

.~ While a particular embodiment of the invention has been shown and described, modifications may be made, and it is intended in the appended c].aims to cover all embodiments which come within the spirit and scope of the invention.

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Claims (11)

WHAT IS CLAIMED IS:

1. A photosynthetically-driven biological fuel cell comprising: means forming a growth chamber having a trans-parent top exposed to sunlight; a nutrient aqueous solution contained in said growth chamber; an anode electrode and a cathode electrode mounted in said growth chamber immersed in said solution in isolated relationship with one another and electrically connected to an external load; a plurality of isolated mesophyll cells suspended in said nutrient aqueous solution, said mesophyll cells producing malate when illuminated by sunlight, said nutrient solution being capable of sustaining the isolated mesophyll cells in an autotropic state and of initiating malate export from the illuminated mesophyll cells into the solution; a selected substance suspended in said nutrient solution to act on the malate to form NADH; and a potential mediator substance suspended in said nutrient solution to transfer electrons from said NADH to said anode.

2. The biological fuel cell defined in claim 1, and which includes a catalyzing substance suspended in the nutrient solution for catalyzing the action of said potential mediator.

3. The biological fuel cell defined in claim 2, and which includes means creating an anaerobic region in the vicinity of said anode to protect the catalyzing step from energy-using oxygen consumption.

4. The biological fuel cell defined in claim 1, in which the isolated mesophyll cells are obtained from a sub-stance selected from a class consisting of CAM plant species and C4 type photosynthetic plant species.

5. The biological fuel cell defined in claim 1, in which said selected substance is malic enzyme.

6. The biological fuel cell defined in claim 1, in which said potential mediator compound is selected from a class consisting of methylene blue, benzyl viologen and methyl viologen.

7. The biological fuel cell defined in claim 2, in which said catalyzing substance is xanthine oxidase.

8. The biological fuel cell defined in claim 3, in which said means creating an anaerobic region comprises a divider member porous to ions and solute molecules but im-pervious to mesophyll cells.

9. The fuel cell defined in claim 3, in which said means creating an anaerobic region comprises means venting the said nutrient solution containing malic acid to a source of vacuum, and means releasing the vacuum with nitrogen gas before circulation to anode.

10. The fuel cell defined in claim 1, and which includes means in said nutrient solution to initiate malate export from the illuminated mesophyll cells to the solution.

11. The fuel cell defined in claim 10, in which said last-named means comprises pyruvic acid at approximately 5m molar (5 X 10-3m) concentration.