Deposition of Metallic Platinum in BlueâGreen Algae Cells - ACS

Chapter 12

Deposition of Metallic Platinum in Blue—Green Algae Cells G.

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Downloaded by NORTH CAROLINA STATE UNIV on October 17, 2012 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch012

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Duncan Hitchens , Tom D. Rogers , Oliver J. Murphy , Comer O. Patterson , and Ralph H. Hearn, Jr. 1

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Lynntech,

Inc., 7610 Eastmark Drive, Suite 105, College Station, TX 77840 Department of Biology, Texas A&M University College Station, TX 77843

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A method for placing metallic platinum in contact with the photosynthetic membranes of the unicellular blue green alga or cyanobacterium Anacystis nidulans (Synechococcus sp.) is described. It was found that cells treated in this way were capable of forming hydrogen gas when illuminated. The deposited platinum particles acted as a catalyst for the generation of hydrogen from photosynthetic light reactions in the absence of an added exogenous electron transfer agent. This exploratory work indicates that electron transfer can occur directly between the membrane-bound Photosystem I and the Pt particles. Electron micrographs of platinum treated algae show deposits of platinum at the surfaces of the internal photosynthetic membranes. The work has long-term implications for the use of cyanobacteria cells for the photoproduction of hydrogen fuel. The innovative aspect of the research has been to demonstrate a technique for placing metallic conductors in direct contact with the membrane structures of microorganisms. This approach can lead, for example, to new types of selective electrochemical biosensors. Hydrogen has long been recognized as a future fuel and energy transfer medium (7,2). Hydrogen is obtained from abundantiy available water; however, a primary source of energy is required to decompose water molecules. Biocatalytic processes are attractive for coupling solar energy to the decomposition of water because photosynthetic organisms already posses light harvesting structures (chlorophyll and accessory pigments) and associated redox capabilities of Photosystems I and II which are capable of carrying out dissociation of water and charge separation. In numerous studies, photosynthetic electron transport in thylakoid membranes isolated from higher plants has been linked to hydrogen production (see reference 3 for a review of all aspects of photobiological hydrogen production). The coupling of photosynthesis to the production of hydrogen was first demonstrated i n cell free

0097-6156/94/0566-0246$08.00/0 © 1994 American Chemical Society

In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Metallic Platinum in Blue-Green

Algae Cells

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systems consisting of isolated spinach chloroplasts (4-6), with bacterial hydrogenase as the catalyst and ferredoxin as the electron transfer mediator (see Figure l a ) . Instability of the hydrogen forming enzyme is a problem encountered i n these systems. Platinum has been introduced as the hydrogen forming catalyst to provide greater stability (7), however, typically methyl viologen is required as the exogenous mediator (see Figure l b ) which itself is unstable in the presence of oxygen. Greenbaum (8-13) has demonstrated the photoproduction 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. It was assumed that the metallic platinum formed was i n 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 Figure l c ) . The inorganic catalyst is relatively stable i n the presence of oxygen and no other exogenous electron transfer mediator is required. Nevertheless systems based on isolated thylakoid membranes have short active lifetimes even when elaborate immobilization procedures are used (14-16). Blue green algae have photosystems that are much more stable than those of chloroplasts and can retain photosynthetic electron transport activities for many months (17,18). This paper presents a new approach to coupling the photosynthetic electron transport activities of the blue green alga Anacystis nidulans to metallic platinum as the hydrogen forming catalyst, without an exogenous electron transfer mediator (79). C e l l G r o w t h a n d P l a t i n u m Treatment Procedures Cells of Anacystis nidulans strain R 2 (Synechococcus sp., P C C 7942) were grown on M e d i u m C s in a continuous culture apparatus described previously (79). After harvesting, the cells were washed and resuspended in 50 m M phosphate buffer (pH 7.1) containing 3 m M M g C l . The platinum deposition step was analogous to that described by Greenbaum for the platinization of thylakoid membranes (8). A solution containing 5.34 m g / m L of hexachloroplatinic acid i n phosphate buffer was neutralized to p H 7 with N a O H . One m L of this solution was combined with 5 m L of cell suspension. The 6-mL volume was placed i n a sealed glass vessel fitted with inlet and outiet ports. The cells were allowed to incubate i n the platinum salt solution in the dark usually for 14 hours. After incubation, the deposition of platinum metal particles was carried out by passing molecular hydrogen (see E q . 1) through the headspace above the sample i n the sealed vessel. 2

Pt(Cl)

2 6

( a q )

+

211^

-->

Pt

(s)

+ 6Cr

(aq)

+ 4H

+ ( a q )

(1)

Typically, hydrogen was passed into the vessel for 30 m. During this time, the color of the cell suspension darkened and metallic platinum (platinum black) particles were visible i n the suspension and adhered to the walls of the vessel. The platinum treated cell suspension was filtered and washed before being used further.


248

ENZYMATIC CONVERSION O F BIOMASS F O R FUELS PRODUCTION


Electron Micrograph Studies of Platinum Treated Cells Electron micrographs ( E M s ) of intact cells of Anacystis that have been platinized are shown in Figures 2 and 3. After filtering and washing, the cells were fixed i n glutaraldehyde-cacodylate solution, embedded i n Spurr's resin and sectioned for E M . The cells were not stained. The optically dense regions are metallic platinum particles. In the frame is one cell that shows metal like particles distributed throughout its internal structure. Figure 2 also shows that not all the cells were platinized to the same degree. Large Pt particles can be seen i n areas corresponding to the bulk solution and adhered to the outer surfaces of one of the cells. Examination of the close-up (i.e., Figure 3) shows the presence of platinum microparticles i n close proximity to the intracellular photosynthetic membranes. Moreover, the deposited platinum particles follow the outline of the thylakoids which, in A. nidulans, appear to be arranged i n concentric shells. Nierzwicki-Bauer et al (20) have shown that i n a marine coccoid cyanobacterium, photosynthetic thylakoids are arranged i n concentric shells. Significantly, these micrographs indicate that the cell membrane and the outer thylakoid layers do not act as a significant permeability barrier limiting the amount of intracellular [Pt(Cl )] " i f a sufficient incubation time in the platinum salt solution is used. Figure 4 was included for comparison purposes. It shows A. nidulans cells that had neither been incubated in the platinum salt solution or had been subject to hydrogen treatment. There were no indications of the presence of metallic platinum i n these cells. 2

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Hydrogen Production Measurements Experiments were conducted to determine i f platinum treated cells could produce hydrogen when illuminated. Measurements of light-dependent hydrogen formation were made amperometrically (21,22). This method is known to be sensitive to l o w aqueous phase hydrogen concentrations and responds rapidly. Before making the measurements, the platinization step was carried out as described except that samples of the cell suspension were withdrawn from the sealed vessel at intervals of 5, 15 and 30 m after starting hydrogen gas flow into the head space. B y varying the exposure to hydrogen, the aim was to vary the amount of metallic platinum deposited i n the cells. T w o identical samples were taken at each time given above. Light-dependent hydrogen evolution was measured using one sample, and photosynthetic oxygen evolution was measured from the other. Hydrogen measurements were carried out i n a thermostated 0.6 m L volume reaction cell (Diamond General, Inc., M o d e l 1271) described i n reference 19. Light from a projector lamp was passed through a cut-off filter (A=above 600 nm) before being directed onto the front of the sample chamber and through an optical window. Prior to each hydrogen measurement, electrode conditioning was carried out by potential cycling between anodic and cathodic limits, typically 40.6 and - 0 . 6 V vs A g / A g C l at 100 m V sec" , for 10 m . Careful choice of working electrode potential was also necessary for hydrogen detection. The sensitivity to hydrogen was measured over a range of potentials by making 50 p L additions of hydrogen-saturated phosphate buffer solution to the reaction cell. The system was most sensitive to hydrogen at 0.35 V vs A g / A g C l which was used subsequently for a l l hydrogen 1


12. H I T C H E N S E T A L .


m

» ( > ujl

/ \

a


HjO^

•

/

H O *

PSII

ao

(c)

PSII

mo

>)

(b)

r

H„0 '

Algae Cells

•

f PSI

Ferredoxin

• Hydrogenase

\

f PSI

/ •» PSII

249

Methyl viologen

-

Platinum

1

\ ^

-

PSI

Platinum

F i g u r e 1. Schemes for Light-Driven Photobiological Hydrogen Formation from Water.

F i g u r e 2. A Transmission Electron Micrograph of Platinized cells of Anacystis nidulans: (magnification x 30,000).



250

ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION

F i g u r e 3. A Transmission Electron Micrograph of a Platinized Anacystis cell (magnification x 96,000).

nidulans

F i g u r e 4. Untreated Anacystis nidulans Cells (magnification x 30,000)



12.

HITCHENS ET AL.


Algae Cells

251

determinations. Above this potential there was a pronounced decrease in sensitivity of the amperometric technique to hydrogen. This is expected since at neutral p H , the onset of oxide film formation on the platinum working electrode surface occurs at around 0.4 V vs A g / A g C l . Platinum oxide is a less catalytic surface for hydrogen oxidation than metallic platinum. Before each run, the system was calibrated with additions of hydrogen-saturated buffer. The response time after injection of hydrogen-saturated buffer was rapid (10-20 s). Variation i n sensitivity to hydrogen was observed between experimental runs. The current was specific to hydrogen since the electrochemical reactant must be a non-ionized small molecule i n order to penetrate the membrane and oxidizable at 0.35 V vs A g / A g C l . The same set-up was routinely used for oxygen evolution measurements except that a working electrode potential of -0.65 V vs A g / A g C l was used. The results of light-dependent hydrogen and oxygen production are given in Table I. The samples of the cell suspension taken immediately before beginning the platinum deposition step (platinum deposition time = 0 m) d i d not produce detectable amounts of hydrogen when illuminated. The sample taken after 5 m of platinum deposition produced measurable quantities of hydrogen. The amount of hydrogen generated increased with duration of the platinum deposition step until reaching a maximum at 30 m. T o continue platinization of the cell suspension beyond 30 m was counter-productive in terms of hydrogen production. Oxygen production decreased in cells that had been subject to platinum deposition. The reduction in oxygen production could have been due to damage to the alga's photosynthetic apparatus by metallic platinum deposits or possibly the presence of the finely dispersed platinum catalyst encourages oxygen and hydrogen recombination to water. It was assumed that since hydrogen formation occurred i n the absence of an exogenous electron transfer agent, 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 as illustrated i n Figure 5 (see also 10). The photosynthetic apparatus of blue green algae is functionally, structurally and molecularly similar to that of chloroplasts from eukaryotic organisms (23-25). Blue green algae have the ability to perform oxygenic photosynthesis and photosynthetic reactions take place in thylakoids. The thylakoids contain two reaction centers (Photosystems I and II) in which the photo-induced electron transfer reactions occur. The two photosystems act i n series and encompass the redox span from H 0 / V 0 to N A D P 7 N A D P H (nicotinamide adenine dinucleotide phosphate). The electron transfer reactions are vectorial i n nature with the reaction centers oriented such that the oxidation of water by PS II occurs on the inner face of the membrane (i.e., facing the thylakoid space) with electrons emerging from PS I on the opposite, or stroma, side of the membrane (26,27). Details of the organization of the integral membrane 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 i n higher plants (28). Still less information is available concerning organization of the PS I complex in cyanobacteria, but published data indicate similarities i n organization between the cyanobacterial complex and that of higher plants (29). In cyanobacteria, the cytoplasmic face of thylakoids corresponds to the stromal face of thylakoids in higher plants. W e therefore assume that some 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. 2

2

2


252


T a b l e I. Hydrogen and Oxygen Formation Rates for Platinum Treated Cells


T i m e used for Pt Deposition (min)

0 5 15 30

Oxygen (nmol h m L 0

Hydrogen (nmol h m L ' )

1

1

1730 1225 1170 1035

0.0 12.3 163.0 655.0

Platinum particle

PS II

H 0-^l/20 +2e-+2H 2

2

LIGHT

LIGHT

F i g u r e 5. Photosynthetic Electron Transport i n Blue Green Algae Showing Metallic Platinum in Electrical Contact with the Reducing Side of PS I (adapted from references 23-25).


12. HITCHENS ET AL.

Metallic Platinum in Blue—Green Algae Cells

253


Table I shows that the highest hydrogen photoproduction rates attained were approximately 25% of the rate expected i f all the electrons derived from the decomposition of water were used for hydrogen formation. In contrast, for platinized thylakoid membranes isolated from higher plants (8), steady state rates of oxygen and hydrogen production indicated that all electrons derived from PS II water splitting were used for hydrogen generation. Hydrogen production may have been limited because not all PS I reducing sites were in electronic contact with a hydrogen forming catalyst particle. The electron micrographs show that only selected cells contained platinum particles which can account for the low hydrogen yields compared to the oxygen yields. S u m m a r y a n d Conclusions Photobiological hydrogen production was achieved indicating that the platinum metal particles are i n direct electronic contact with PS I. Whether this system offers the advantage of stable light-driven hydrogen production over long periods of time remains to be determined. In addition, many breakthroughs are required (3) to make photobiological hydrogen production systems both technically and economically viable as a means of fuel generation e.g., for hydrogen powered vehicles (30). The present approach has the cost disadvantage of requiring a noble metal catalyst. The work has, however, demonstrated a first step towards a viable means of making electrical contact to the charge transfer complexes contained within cells, particularly the redox complexes associated with membrane energy transduction. Usually, freely diffusing redox mediators are needed to relay electrons indirectly between the redox centers of proteins and electrodes (57). Methods that enable direct electrical contact between biological molecules and electrodes are being developed at a fast pace (32-34); however, these methods have largely focused on soluble redox proteins and enzymes. Direct electrochemical studies of intact multi-enzyme structures associated with photosynthetic (and respiratory) membranes is a new challenge. The technique described i n this paper may have some long-term relevance to this problem. Being able to make direct electrical contact to membrane proteins can have practical benefits in several areas including: selective biosensors (35,36), biomolecular devices (10), and biofuel cells (37). Acknowledgment W e wish to thank the National Science Foundation (Small Business Innovation Research A w a r d ISI-8961216) for support of this work. References

1. Bockris, J. O ' M . Energy Options; Australia & New Zealand Book Company: Sydney, Australia, 1980. 2. Gutmann, F.; Murphy, O.J. In Modern Aspects of Electrochemistry; Bockris J. O ' M ; White R.E.; Conway B.E., Eds.; Plenum Press: New York, N Y , 1983, Vol. 15; p 1. 3. Weaver, P.F.; Lien, S.; Seibert, M . Solar Energy 1980, 24, 3. 4. Arnon, D.; Mitsui, A.; Peneque, A . Science 1961, 134, 1425.


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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37.


Beneman, J.R.; Berenson, J.A.; Kaplan, N.O.; Kamen, D. Proc. Nat. Acad. Sci. U.S.A. 1973, 70, 2317. Greenbaum, E. Biotechnol. Bioeng. Symp. 1980, 10, 1. Gisby, P.E.; Hall, D.O. Nature 1980, 287, 251. Greenbaum, E. Science 1985, 230, 1373. Greenbaum, E. J. Phys. Chem. 1988, 92, 4571. Greenbaum, E. In Molecular Electronic Devices; Carter, F.L.; Siatkowski, R.E.; Wohltjen, H., Eds.; Elsevier Science Publishers B.V.: North Holland, 1988; p 575. Greenbaum, E. Bioelectrochem. Bioenerg. 1989, 21, 171. Greenbaum, E. J. Phys. Chem. 1990, 94, 6151. Greenbaum, E. J. Phys Chem. 1992, 96, 514. Roa, K.K.; Rosa, L.; Hall, D.O. Biochem. Biophys. Res. Comm. 1976, 68, 21. Woodward, J.; Greenbaum, E. Biotechnology and Bioengineering Symp. 1983, 13, 271. Coquempot, M.F.; Thomasset, B.; Barbotin, N.J.; Gellf, G.; Thomas, D. Eur. J. Appl. Microbiol. Biotechnol. 1981, 11, 193. Affolter, D.; Hall, D.O. Photobiochem. Photobiophys. 1986, 12, 193. Gisby, P.E.; Rao, K.K.; Hall, D.O. Method. Enzymol. 1987, 135, 440. Hitchens, G.D.; Rogers, T.D.; Murphy, O.J.; Patterson, C.O. Biochim. Biophys. Res. Comm. 1991, 175, 1029. Nierzwicki-Bauer, S.A.; Balkwill, D.L.; Stevens, S.E. Jr. J. Cell Biol. 1983, 97, 713. 21.Wang, R.; Healey, H.L.; Myers, J. Plant Physiol. 1971, 48, 108. Jones, L.W.; Bishop, N.I. Plant Physiol. 1976, 57, 659. Cramer, W.A.; Crofts, A.R. In Photosynthetic Energy Conversion in Plants and Bacteria; Govindjee, Ed.; Academic Press: New York, N Y , 1982; p 387. Stewart, A.C.; Bendall, D.S. Biochem. J. 1981, 194, 877. Guikema, J.; Sherman, L . Biochim. Biophys. Acta, 1982, 440. Harold, F . M . The Vital Force: A Study of Bioenergetics; W.H. Freeman: New York, N Y , 1986. Topics in Photosynthesis; Barber, J., Ed.; Elsevier: Amsterdam, Holland, 1987, Vol. 8. Ortiz, W.; Lam, E.; Chollar, S.; Munt, D.; Malkin, R. Plant Physiol. 1985, 77, 389. Wynn, R.M.; Omaha, J.; Malkin, R. Biochemistry 1989, 28, 5554. Lemons, R.A. J. Power Sources 1990, 29, 251. Turner, A.P.F. In Analytical Uses of Immobilized Biological Compounds for Detection, Medical and Industrial Uses; Guilbault, G.G.; Mascini, M . , Eds.; Reidel Publishing Co.: Boston, M A , 1988; p 131. Armstrong, F.A.; Cox, P.A.; Hill, H.A.O.; Lowe, V.J.; Oliver, B.N. J. Electroanal. Chem. 1987, 217, 331. Frew, J.E.; Hill, H.A.O. Eur. J. Biochem. 1988, 172, 261. Hitchens, G.D. Trends Biochem. Sci. 1989, 14, 152. Biosensors: Fundamentals and Applications; Turner, A.P.F.; Karube, I; Wilson, G.S., Eds.; Oxford University Press: Oxford, Great Britain, 1987. Yacynych, A . M . In Bioinstrumentation, Research Developments and Applications; Wise, D.L., Ed.; Butterworth Publishers: Stoneham, M A , 1990; p 1317. Bennetto, H.P. Life Chemistry Reports; 1984, 2, 303.

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Deposition of Metallic Platinum in BlueâGreen Algae Cells - ACS

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