A new photocatalytic material based on algal cells

Vol. 175, No. 3, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages 1029-l 035

March 29, 1991

A NEW PHOTOCATALYTIC ALGAL

MATERIAL CELLS

BASED ON

G. Duncan Hitchens* , Tom D. Rogers and Oliver J. Murphy Lynntech, Inc. 111 East 27th Street., Suite 204 Bryan, Texas 77803 and Comer 0. Patterson Departmentof Biology Texas A & M University, College Station, Texas 77843-2891 Received

February

22,

1991

Metallic platinum was deposited at surfaces of intracellular photosynthetic membranesof whole cells of a cyanobacterium(blue-greenalga). The depositedplatinum particlesacted asa catalyst for generationof hydrogen from photosynthetic decomposition of water in the absenceof other exogenous electron transfer agents. This technique representsa meansof placing metal catalysts in contact with intracellular structures of microorganisms. 0 1991 Academic Press, Inc.

This report describesthe useof platinum-treated cells of the unicellular blue green alga or cyanobacteriumAnucystis nidulam (Synechococcussp.) asa novel systemfor the decompositionof water into oxygen and hydrogen fuel using solar energy. Hydrogen has long been recognized as a future fuel and energy transfer medium ]1,2]. Hydrogen is obtainedfrom abundantly available water: however, a primary sourceof energy is required to decomposewater molecules. Biocatalytic processesare attractive for coupling solar energy to the decompositionof water becausephotosynthetic organismsalready posses light harvesting structures (chlorophyll and accessory pigments) and associatedredox capabilities of PhotosystemsI and II which are capable of carrying out dissociation of water and chargeseparation. In numerousstudies, photosynthetic electron transport in thylakoid membranes isolated from higher plants has been linked to hydrogen production ]see e.g. 3-51. Instability of the hydrogen forming catalyst hydrogenase, and oxygen sensitivity of exogenouselectron transfer intermediates, such as methyl viologen, are problems often encounteredin thesesystems.Recently, Greenbaum161demonstratedthe photoproduction * To whom correspondenceshouldbe addressed. 0006-291X/91

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Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of hydrogen and oxygen using a platinum catalyst deposited directly onto isolated thylakoid membranes. Platinum was deposited from a dissolved platinum salt using hydrogen as the reductant a-ding to the following reaction: ~(C1)6~-(ti

+

2H2cg)

+

ptcs>+ Kl-(ac~ + 4H+(~

It was assumed that the metallic platinum formed was in direct contact with the reducing side of Photosystem I in such a way that electron transfer could occur directly between the membrane-bound Photosystem I and the platinum [see also 7-91. The advantages offered by this approach are as follows: (i) the inorganic catalyst (platinum) is relatively stable in the presence of oxygen; and, (ii) no other exogenous electron transfer agent is required. Nevertheless, the short active life time of photosynthetic electron transport in isolated thylakoids is a concern. Essentially the same photosynthetic apparatus is present in thylakoids of cyan-a. Intact cyanobacteria cells ate quite rugged and photosynthetic electron transport activity can be maintained almost indefinitely in immobilized cells [lo]. Hydrogen production from appropriately treated cyanobacterial cells therefore offers the promise of commercially important applications. This paper describes preliminary research into a method for depositing platinum catalyst particles in direct contact with the intracellular photosynthetic membranes of whole cells of a cyanobactium, and reports measurements of hydrogen production from cells treated in this way. MATERIALS Growth

AND METHODS

of Cells

Cells of Anacystisnidulans strain R2 (Synechococcus sp., PCC 7942) were grown on Medium Cs [ 1 l] in a continuous culture apparatus at 33oC. The cell suspension was stirred and CO2 was provided by constantly bubbling the culture with a gas mixture consisting of 1% CO2 in air. The culture was illuminated by two 40W tungsten incandescent lamps. This produced a relatively low light intensity of 66 FE m-2 s-1 at the outside surface of the culture vessel. After harvesting, the cells were washed and resuspended in 50 mM phosphate buffer (pH=7.1) containing 3 mM MgQ. Hydrogen

and Oxygen Measurements

Measurements of light-dependent hydrogen formation were made amperometrically [12,13]. This method is known to be sensitive to low aqueous phase hydrogen concentrations and responds rapidly. Measurements were carried out in a thermostated 0.6 ml volume reaction cell (Diamond General, Inc., Model 1271) incorporating a miniature Clark electrode assembly consisting of a platinum sensing electrode (diamebz 125 p.m) and a Ag/AgCl reference electrode (Diamond General, Inc., Model 730). The cell also included a gold counter electrode, magnetic stirrer and stirrer assembly. Electrochemical measurements were made using a three electrode technique. A PAR 362 potentiostat was used to polarize the platinum working electrode and the resulting currents were displayed on a Linear X-t chart recorder. The platinum and Ag/AgCl electrode were sealed using a membrane supplied by Yellow Springs Instrument Co. The same set-up was routine1 used for oxygen evolution measurements. Lightfiomaprojectorlampwaspassedthrou $: a cut-off filter (Labove 600 nm) before being directed onto the front of the sample Chamber.

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Prior to each hydrogen measurement,electrode conditioning was carried out by potential cycling betweenanodic andcathodic limits, typically +0.6 and -0.6V vs Ag/AgCl at 100 mV secW1,for 10 minutes. The membranewas replaced at least once per day. Careful choice of working electrodepotential was alsonecessaryfor hydrogen detection as shownin Figure I. At eachpotential, the systemwasallowed to stabilizeafter which 50 ~1 of hydrogen-saturatedphosphatebuffer wasinjected into the cell. An electrodepotential of 0.35 V vs Ag/AgCl was usedfor all hydrogen determinations. Above this potential there was a pronounceddecreasein sensitivity of the amperometrictechnique. This is expected sinceat neutral pH, the onset of oxide film formation on the platinum working electrode surfaceoccurs at around 0.4 V vs Ag/AgCl. Platinum oxide is a lesscatalytic surfacefor hydrogen oxidation than metallicplatinum. Before each run, the systemwas calibrated with additions of hydrogen-saturated buffer. The responsetime after injection of hydrogen-saturatedbuffer was rapid (lo-20 seconds). Variation in sensitivity to hydrogen was observedbetweenexperimental runs. The current was specific to hydrogen since the electrochemical reactant must be a nonionized smallmoleculein order to penetratethe membraneand oxidizable at 0.35 V vs AgIAgCl.

RESULTS ANDDISCUSSION A procedure for was developed for the deposition of metallic platinum onto photosynthetic membranes(thylakoids) of A. nidulans. The procedure is based on the method describedpreviously [6]. A solution containing 5.34 mg/ml of hexachloroplatinic acid in phosphatebuffer was neutralizedto pH=7 with NaOH. One ml of this solution was combined with 5 ml of cell suspensioncontaining 34.5 mg of chlorophyll. The 6 ml volume wasplaced in a seatedglassvesselfitted with inlet and outlet ports. It wasfound helpful to incubatethe cell suspensionin the platinizing solutionfor at least 14hoursbefore proceedingwith the depositionstep.

Deposition of platinum metal particles was carried out by passing molecular hydrogen through the headspaceabove the sample in the sealedvessel. At intervals, hydrogen gas flow was stopped and 0.6 ml aliquots of the hydrogen-treated cells were removed from the sealed vessel and transferred to the electrochemical cell for

0.0

0.2 POLARIZING

0.4 POTENTIAL

0.6 (YS AglAgCI)

0.6

FIGURE 1. Amperometric hydrogen detection as a function of electrode potential. Conditions are given in the materials and methods section.

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2ooo J ( 1500

+

OXYGEN

+

HYDROGEN

-

I I 0

5

10

DURATION

15

20

OF PLATlNlZATlON

25

30

35

(minutes)

I

02

I

I

I

I

4 6 8 TIME (MINUTES)

I

I

10

12

FIGURE2. Ratesof hydrogenand oxygenphotoproduction by Anacystis niddam cells asa functionof platinizationduration.Measurements of hydrogenandoxygenproduction weremadeon cell samples takenfrom sealedplatinizationvesselat varioustimeintervats followingprocedures givenin thetext. FIGURE3. Tracingsof recorderdatacomparinghydrogenandoxygenphotoproduction by Amcysti nidu.4zn.r cellsafter 30minutesof platinizationwith molecularhydrogen.The arrowspointingupanddownindicatelight onandoff respectively. measurements of hydrogen production. Cell transferswere carried out under

nitrogen. Cell

sampleswere flushed with nitrogen to remove dissolved hydrogen before beginning measurementsof hydrogen production. A secondsample(0.6 ml) was taken at the same time to obtain complementarydata on photosynthetic oxygen production by the platinized cells. Roth sampleswere discardedafter measurements were made,and platinization of the cell suspensionin the sealedvesselwasresumedby restarting hydrogen flow through the headspace.

Figure 2 showshydrogen and oxygen formation rates after various durations of platinization in the sealedvessel. After

15 minutesof hydrogen treatment in the sealed

vessel, there was an increasein the light-dependent current (correspondingto hydrogen production) during measurementin the electrochemicalcell. The highestlight-dependent current (highestrate of hydrogen production) wasachievedafter approximately 30 minutes of platinization. Figure 3 showstracings of the recorder data for hydrogen and oxygen photoproduction after 30 minutesof platinization. F.&her exposureto hydrogen beyond 30 minutes failed to give increasedresponseduring the electrochemical measurementof hydrogen production. In experimentsin which the platinum salt solution was treated with hydrogen first, followed by addition of the cells, no light-dependent hydrogen formation wasdetected. No other electron transfer agent, suchas methyl viologen, wasaddedto the cells. Ferredoxin and NADPH do not readily undergo electron exchange with metal catalysts. Therefore, it appearsthat ionic platinum was reduced to form colloidal metal platinum or platinum isletsin such a way that they directly contact the reducing siteof PS I to form a novel photocatalytic system. Detailsof the organizationof the integral membrane 1032

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components making up PS I core complex are still unclear, but it appears that several component polypeptides are exposed on the stromal face of the thylakoid in higher plants [ 141. Still less information cyanobacteria,

is available concerning organization of the PS I complex in

but published

data indicate similarities

in organization

between

the

cyanobacterial complex and that of higher plants 1151. In cyanobacteria, the cytoplasmic face of thylakoids corresponds to the stromal face of thylakoids in higher plants. We therefore assume that metallic platinum particles are deposited on the cytoplasmic faces of cyanobacterial thylakoids in immediate proximity to surface-exposed components of the PS I core complex.

Figure 2 shows that the highest hydrogen photoproduction

rates attained were

approximately 25% of the rate expected if all the electrons derived from the decomposition In contrast, for platinized thylakoid were used for hydrogen formation.

of water

membranes isolated from higher plants 161, steady state rates of oxygen and hydrogen production indicated that all electrons derived from PS II water splitting were used for hydrogen generation. Several factors may contribute to the decreased flow of electrons to hydrogen production

in our cells.

First, hydrogen production may have been limited

because not all PS I reducing sites were in electronic contact with a hydrogen forming catalyst particle. The cell membrane may act as a permeability barrier limiting the amount of intracellular IPt(Cl,)]2-. Nierzwicki-Bauer et al [ 16) have shown that in a marine coccoid cyanobacterium, photosynthetic thylakoids are arranged in concentric shells. The outer thylakoid layers may act as permeability barriers, limiting access of platinum ions to the inner thylakoid layers. Thylakoids in A. niduluns appear to be arranged in the same pattern (C.O. Patterson unpublished). Thus, there may be several barriers limiting access of platinum to PS I sites in thylakoids. Experiments were conducted aimed at increasing the amount of [Pt(Cl,)12- within cells prior to the deposition step. In one set of experiments, cells were subjected to freeze-thaw cycles to increase cell membrane permeability to [Pt(Cl,)12-. In another set of experiments, 3% v/v toluene was included in the platinizing solutions. Toluene treatment has been used to passively equilibrate intracellular contents of bacterial cells with a given extracellular concentration of a small molecules without

greatly affecting protein-protein

interactions of membranes [ 17,181.

Neither method resulted in improved hydrogen yields.

It is likely that the complete PS 1 electron transfer pathway from plastocyanine to NADP+ (and ultimately to CO.9 remains essentially intact and operating in illuminated platinized whole cells. Normal endogenous electron acceptors of PS I may compete for electrons with the platinum catalyst, thereby, keeping the hydrogen yield relatively low. Placement of metallic platinum particles directly adjacent to the reducing side of PS I is crucial for hydrogen photoproduction from whole cells. This is because electron transfer rates between donor and acceptor centers in biological systems decay exponentially with 1033

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distance between the centers involved [see e.g., 19,201 according to the following relationship: k=ead where k is the electron transfer rate, a is a factor that is medium-dependentand d is the distancebetween the centers. In proteins, electron transfer ratesdrop by a factor of about 104when distancebetweenelectron donor and acceptorcentersis increasedfrom 8 to 17A [20]. In our platinized whole cells, ferredoxin, the immediateelectron acceptor for PS I, may remainassociatedwith a significant traction of the PS I complexesin the cell. Bound ferredoxin can prevent close interaction betweenthe reducing sideof PS I and [Pt(C1,)]2resulting in large electron transfer distancesbetweenPSI and the depositedmetalparticles. During the 1Chour incubationof the cells in the platinizing solution,somedisruptionof the normalprotein-protein interactionsof the PS I reaction centercomplex may occur, allowing closer interaction between [Pt(Cl,)]*- and PS I prior to deposition. When isolatedhigher plant thylakoids were platinized [6], ferredoxin was dissociated during membrane preparation. This aided hydrogen production by decreasingthe electron transfer distances betweendepositedmetallic platinum particlesand the PS I reducing site.

Studiesof the structureof platinum metalcatalystsindicatethat maximumactivity is obtainedfrom colloidal particlesapproximately 20-30 A in diametermadeup of around500 atoms 121,221. It is possible that the intracellular environment associated with photosyntheticmembranescan imposesteric constrainson the platinumparticles during the precipitation step, preventing them from attaining optimal size for full catalytic activity insidethe cells.

In summary, biophotocatalytic hydrogen production from water was achieved without using highly oxygen sensitive components. With appropriate immobilization procedures, cells of this type are capable of stable photosynthetic activity over many Hence, this approach has the potential for sustained hydrogen months [IO]. photoproduction over longer periods than can be achieved with isolated thylakoid membranes.It may be possibleto usethis techniquefor the depositionof metallic catalysts other than platinum. ACKNOWLEIXGMENTS We wish to thank the National Science Foundation (Small BusinessInnovation ResearchAward 8961216)for supportof this work and CarlosE. Salinasfor many helpful commentsand suggestions. REFERENCES 1.

Bockris, J. OM Sydney.

(1980) Energy Options, Australia & New Zealand Book Co.,

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Gutmann, F., and Murphy, O.J. (1983) In Modern Aspects of Electrochemistry, (J. O’M. Bock+ R. E. White and B.E. Conway, Eds.) Vol 15, pp l-82 Plenum Press, New York. Beneman, J.R., Berenson, J.A., Kaplan, N.O., Kamen D. ( 1973) Proc. Nat. Acad. Sci. U.S.A. 70, 2317-2329. Weaver, P.F., Lien, S., and Seibert, M. (1980) Solar Energy 24, 3-45. Gisby, P.E., and Hall, D.O. (1980) Nature 287, 251-253. Greenbaum, E. (1985) Science 230, 1373-1375. Greenbaum, E. (1988) J. Phys. Chem. 92,4571-4576. Greenbaum, E. ( 1989) Bioelectrochem. Bioenerg. 2 1, 17 1- 177. Greenbaum, E. (1990) J. Phys. Chem. 94, 6151-6153. Affolter, D., and Hall, D.O. (1986) Photobiochem. Photobiophys. 12, 193-201. Stevens, Jr. S.E., Patterson C.O., and Myers, J. (1973) J. Phycol. 9, 427-430. Wang, R., Healey F. P., and Myers J. (1971) Plant Physiol. 48, 108-l 10. Jones, L.W., and Bishop, N.I. (1976) Plant Physiol. 57,659-665. Ortiz, W., Lam, E.. Chollar, S., Mum, D., and Malkin, R. (1985) Plant Physiol. 77, 389-397. Wynn, R.M., Omaha, J., and Malkin, R. (1989) Biochemistry 28,5554-5560. Nierzwicki-Bauer. S.A., Balkwill, D.L. and Stevens, Jr., S.E. (1983) J. Cell Biol. 97, 713-722. Gachelin, G., (1969) Biochem. Biophys. Res. Commun. 34,382-387. Booth, I.R., and Morris, J.G. (1982) Bioscience Reports 2,47-53. McLendon, G., Miller, J.R., Simolo, K., Taylor, K., Mauk, A.G., and English, A.M., (1986) In Excited States and Reactive Intermediates (A.B.P. Lever Ed.) pp 150-165, American Chemical Society, Washington D.C. Mayo, S.L., Ellis W.R., Crutchley R.J., and Grey, H.B. (1986) Science 233, 948-952. Toshima, N., Kuriyama, M., Yamada, Y., Hirai, I-I. (1981) Chem. L.&t. 793-796. Kiwi, J., and Gratzel, M.J. (1984) J. Phys. Chem. 88, 1302-1307.

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