Geranium Leaf Assisted Biosynthesis of Silver Nanoparticles

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ARTICLES Geranium Leaf Assisted Biosynthesis of Silver Nanoparticles S. Shiv Shankar,† Absar Ahmad,*,‡ and Murali Sastry*,† Materials Chemistry and Biochemical Sciences Divisions, National Chemical Laboratory, Pune 411 008, India

Development of biologically inspired experimental processes for the synthesis of nanoparticles is evolving into an important branch of nanotechnology. In this paper, we report on the use of Geranium (Pelargonium graveolens) leaf extract in the extracellular synthesis of silver nanoparticles. On treating aqueous silver nitrate solution with geranium leaf extract, rapid reduction of the silver ions is observed leading to the formation of highly stable, crystalline silver nanoparticles in solution. Transmission electron microscopy analysis of the silver particles indicated that they ranged in size from 16 to 40 nm and were assembled in solution into quasilinear superstructures. The rate of reduction of the silver ions by the geranium leaf extract is faster than that observed by us in an earlier study using a fungus, Fusarium oxysporum, thus highlighting the possibility that nanoparticle biosynthesis methodologies will achieve rates of synthesis comparable to those of chemical methods. This study also represents an important advance in the use of plants over microorganisms in the biosynthesis of metal nanoparticles.

Introduction An important area of research in nanotechnology concerns the synthesis of nanoparticles of different chemical compositions, sizes, shapes, and controlled polydispersity. Currently, there is a growing need to develop environmentally benign nanoparticle synthesis processes that do not use toxic chemicals in the synthesis protocol. As a result, researchers in the field of nanoparticle synthesis and assembly have turned to biological systems for inspiration. This is not surprising given that many organisms, both unicellular and multicellular, are known to produce inorganic materials either intra- (1) or extracellularly (2). Some well-known examples of the synthesis of inorganic materials by bio-organisms include magnetotactic bacteria (which synthesize magnetite nanoparticles) (3-5), diatoms (which synthesize siliceous materials) (6-8), and S-layer bacteria (which produce gypsum and calcium carbonate layers) (9, 10). The secrets gleaned from nature have lead to the development of biomimetic approaches for the growth of advanced nanomaterials. Even though many biotechnological applications such as remediation of toxic metals employ microorganisms such as bacteria (11) and yeast (12) (the detoxification often occurring via reduction of the metal ions/formation of metal sulfides), it is only relatively recently that materials scientists have been viewing with interest such microorganisms as possible ecofriendly nanofactories (13-18). Beveridge and co-workers have demonstrated * To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. † Materials Chemistry Division. ‡ Biochemical Sciences Division. 10.1021/bp034070w CCC: $25.00

that gold particles of nanoscale dimensions may be readily precipitated within bacterial cells by incubation of the cells with Au3+ ions (13-15). Klaus-Joerger and co-workers have shown that the bacterium Pseudomonas stutzeri AG259, isolated from a silver mine, when placed in a concentrated aqueous solution of AgNO3, resulted in the reduction of the Ag+ ions and formation of silver nanoparticles of well-defined size and distinct morphology within the periplasmic space of the bacteria (16-18). Nair and Pradeep have synthesized nanocrystals of gold, silver, and their alloys by reaction of the corresponding metal ions within cells of lactic acid bacteria present in buttermilk (19). In a break from tradition, which has hitherto relied on the use of prokaryotes such as bacteria in the intracellular synthesis of nanoparticles, we have recently shown in this laboratory that eukaryotic organisms such as fungi may be used to grow nanoparticles of different chemical compositions and sizes. A number of different genera of fungi have been investigated in this effort, and it has been shown that fungi are extremely good candidates in the synthesis of gold (20, 21), silver (22, 23), and indeed quantum dots of the technologically important CdS by enzymatic processes (24). We have recently reported that the alkalothermophilic (extremophilic) actinomycete Thermomonospora sp. can synthesize extracellularly high concentration of gold nanoparticles of 8 nm average size with good monodispersity (25). As can be seen from the above, the use of biological microorganisms in the deliberate and controlled synthesis of nanoparticles is a relatively new and exciting area of research with considerable potential for development. While microorganisms such as bacteria, actinomycetes, and fungi continue to be investigated in metal nanoparticle synthesis, the use of parts of whole plants in similar nanoparticle biosynthesis methodologies is an exciting

© 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/01/2003

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possibility that is relatively unexplored and underexploited. Recently, Jose-Yacaman and co-workers demonstrated the synthesis of gold nanoparticles within live alfalfa plants by gold uptake from solid media (26). These authors have reported that Au(III) ions are reduced in the solid media to Au(0) by the plant and then the metal atoms are absorbed into the plant where nucleation and growth of gold nanoparticles takes place. This group has also shown that assemblies of silver nanoparticles may be synthesized within alfalfa shoots by reaction of the plants with silver ions (27). In a related report, agricultural biomass has been used to reduce Cr(VI) to Cr(III) ions (28), indicating that biological methods can be very efficient in decontaminating polluted waters and soil polluted with heavy metal ions. In this paper, we report on the synthesis of silver nanoparticles by the reduction of aqueous Ag+ ions by the extract of geranium leaves (Pelargonium graveolens). An elaborate screening process involving a number of plant species has led us to geranium leaves as a promising candidate for the synthesis of silver nanoparticles. The reduction of the metal ions is fairly rapid, occurs readily in solution, and results in a high density of extremely stable silver nanoparticles in the size range 16-40 nm with an average size ca. 27 nm. The silver nanoparticles appear to be assembled into open, quasilinear superstructures and are predominantly spherical in shape. Presented below are details of the investigation.

Figure 1. UV-vis spectra recorded as a function of time of reaction of 10-3 M aqueous solution of silver nitrate with P. graveolens leaf broth. The time of reaction is indicated next to the respective curves.

Experimental Section The broth used for reduction of Ag+ ions to Ag0 was prepared by taking 20 g of thoroughly washed and finely cut P. graveolens leaves in a 500 mL Erlenmeyer flask with 100 mL of sterile distilled water and then boiling the mixture for 1 min. After boiling, the solution was decanted, and 5 mL of this broth was added to 100 mL of 10-3 M aqueous AgNO3 solution. The bioreduction of the Ag+ ions in solutions was monitored by periodic sampling of aliquots (1 mL) of the aqueous component after 20 times dilution and measuring the UV-vis spectra of the solution. UV-vis spectra of these aliquots were monitored as a function of time of reaction on a Hewlett-Packard diode array spectrophotometer (model HP-8452) operated at a resolution of 2 nm. X-ray diffraction (XRD) measurements of the bioreduced silver nitrate solution drop-coated on glass substrate were carried out on a Phillips PW 1830 instrument operating at a voltage of 40 kV and a current of 30 mA with Cu KR radiation. For Fourier transform infrared (FTIR) spectroscopy measurements, drop-coated samples on Si(111) wafers were prepared in the following manner: 20 mL of bioreduced silver nitrate solution after 24 h of reaction with the leaf broth was centrifuged at 10000 rpm for 15 min, following which the pellet was redispersed in 20 mL of sterile distilled water to get rid of any free proteins/ enzyme molecules that are not bound to the silver nanoparticles. The process of centrifugation and redispersion in sterile distilled water was repeated three times to ensure better separation of free proteins/enzymes from the silver nanoparticles. FTIR measurement was carried out on a Perkin-Elmer instrument in the diffuse reflectance mode at a resolution of 4 cm-1. Samples for transmission electron microscopy (TEM) analysis were prepared by drop-coating biosynthesized silver nanoparticle solution (24 h reaction of the silver nitrate solution with the geranium leaf broth) on carbon-coated copper TEM grids. The films on the TEM grids were allowed to stand for 2 min, following which the extra solution was removed using a blotting paper and the grid was allowed

to dry prior to measurement. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage at 120 kV.

Results and Discussion Reduction of the aqueous Ag+ ions during exposure to the broth of boiled P. graveolens leaves may be easily followed by UV-vis spectroscopy. It is well-known that silver nanoparticles exhibit yellowish-brown color in water; this color arises due to excitation of surface plasmon vibrations in the metal nanoparticles (29). Figure 1 shows the UV-vis spectra recorded from the aqueous silver nitrate-geranium leaf broth reaction medium as a function of time of reaction. It is observed that the silver surface plasmon resonance band occurs at ca. 440 nm and steadily increases in intensity as a function of time of reaction without any shift in the peak wavelength. In addition to the peak at 440 nm, another peak at 370 nm is also seen that increases in intensity with time and appears as a shoulder in the UV-vis spectra after 4 h of reaction. We believe this shoulder at 370 nm corresponds to the transverse plasmon vibration in the silver nanoparticles whereas the peak at 440 nm is due to excitation of longitudinal plasmon vibrations. That these wavelengths are distinct and separated by 70 nm indicates that the silver nanoparticles in solution are assembled into open, quasilinear superstructures. The reduction of the silver ions occurs fairly rapidly, more than 90% of which is complete within 9 h of reaction (Figure 1). In earlier studies on the synthesis of silver nanoparticles using bacteria (16-19) or fungi (22, 23), the time required for completion of the reaction (i.e., complete reduction of the metal ions) ranged from 24 to 120 h and is thus rather slow. This is one disadvantage of the biosynthetic procedures that needs to be addressed if they are to compete with chemical methods for nanoparticle synthesis. The reduction in reaction time observed for the extract of P. graveolens leaves is a significant advance in this direction. The silver particles

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Figure 2. XRD pattern of silver nanoparticles synthesized using P. graveolens leaf broth. The inset represents the XRD pattern of plain P. graveolens leaf broth.

were observed to be extremely stable in solution even 4 weeks after their synthesis. There has been a recent report by Ping Yong and co-workers on the accelerated synthesis of palladium nanoparticles using the sulfatereducing bacterium Desulfovibrio desulfuricans (NCIMB 8307) (30) wherein the reduction of the metal ions was achieved in a very short time (3 min) (30). However, the process was not purely biosynthetic and depended on an exogenous electron donor to accomplish reduction of the metal ions (20). Furthermore, the Pd nanoparticles were not formed in solution and were adsorbed on the surface of the microorganism (30). Figure 2 shows the XRD pattern obtained for silver nanoparticles synthesized using the geranium leaf broth. A number of Bragg reflections are observed that may be indexed on the basis of the fcc structure of silver. The XRD pattern thus clearly shows that the silver nanoparticles formed by the reduction of Ag+ ions by the geranium leaf extract are crystalline in nature. In addition to the Bragg peaks representative of fcc silver nanocrystals, additional and as yet unassigned peaks (marked by stars) are also observed. In the inset of Figure 2, the XRD pattern of a film cast from the P. graveolens leaf broth alone is shown. Observation of sharp Bragg peaks suggests the presence of a crystalline phase of some bio-organic compounds/protein(s) that are present in the P. graveolens leaf broth. The intensity of the Bragg reflections suggests strong X-ray scattering centers in the crystalline phase and could possibly arise from metalloproteins in the broth. A distinct possibility is the presence of a crystalline phase of the metalloprotein chlorophyll that has a Mg ion within the protein scaffold. Preliminary energy dispersive analysis of X-rays (EDAX) measurements carried out on films of the geranium leaf broth clearly showed the presence of X-ray peaks characteristic of Mg supporting the contention that the sharp Bragg reflections in the XRD pattern could be due to chlorophyll. As mentioned in the Experimental Section, the silver nanoparticles after their formation were repeatedly centrifuged and redispersed in sterile distilled water prior to XRD and TEM analysis, thus ruling out the presence of any free compound/protein that might independently crystallize and give rise to the observed Bragg reflections. The XRD results thus suggest that the crystallization of the bioorganic phase occurs on the surface of the silver nanoparticles (or vice versa). Further work is currently

Figure 3. FTIR spectra of (a) plain P. graveolens leaf broth and (b) capped silver nanoparticles synthesized using P. graveolens leaf broth.

in progress to unambiguously identify this crystalline phase coexisting with the silver nanocrystals. FTIR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag+ ions and capping of the bioreduced silver nanoparticles synthesized by the leaf broth. The silver nitrate solution after complete reduction of Ag+ ions and formation of gold nanoparticles was centrifuged at 10000 rpm for 15 min to isolate the silver nanoparticles from free proteins or other compounds present in the solution, and the centrifugate wascollected for FTIR analysis. Curve (a) of Figure 3 represents the FTIR spectrum of the plain P. graveolens leaf broth and shows peaks at 1736, 1640, and 1458 cm-1. The peak at 1640 cm-1 is assigned to the amide I band of proteins (possibly chlorophyll) released by the geranium leaves (31, 32). The peak at 1736 cm-1 possibly arises from ester CdO groups of chlorophyll (33), while the peak at 1458 cm-1 may be assigned to symmetric stretching vibrations of -COO- (carboxylate ion) groups of amino acid residues with free carboxylate groups in the protein. Curve (b) of Figure 3 represents the FTIR spectrum of silver nanoparticles biosynthesized using the geranium leaf broth. This spectrum exhibits peaks at 1748 and 1640 cm-1. As mentioned above, the peak at 1748 cm-1 may be assigned to ester CdO groups of chlorophyll (33). It is well-known that water-soluble fractions of geranium leaves contain large amounts of terpenoids of which citronellol and geraniol form the major component and linalool a smaller fraction (34). It is possible that the terpenoids also contribute to the reduction of the silver ions and, in the process, are oxidized to carbonyl groups that thus result in a band at 1748 cm-1. On formation of silver nanoparticles, the peak corresponding to the amide I band at 1640 cm-1 has broadened and indicates capping of the silver nanoparticles by the protein (Figure 3, curve b). It is well-known that proteins can bind to gold nanoparticles either through free amine groups or cysteine residues in the proteins (35). A similar mechanism could be operating

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observed may be driven by the growth of crystals of the bioorganic phase inferred from the XRD studies in the space between the silver nanoparticles. Although the TEM pictures do not provide direct evidence of the presence of a bioorganic crystalline phase on the silver nanoparticles, it is interesting to note that most of the particles in the TEM pictures are not in physical contact but are separated by a fairly uniform interparticle distance. From TEM pictures obtained at higher magnification (Figure 4d) it is observed that a second material coats the silver nanoparticles. We believe that this coating material with an average thickness of 5 nm is a bioorganic component of the P. graveolens leaf broth (possibly chlorophyll) and forms a crystalline shell around the silver nanoparticles, giving rise to sharp Bragg reflections. It is known that some amino acids and proteins such as silk fibroin have (through their tyrosine residue) the tendency to reduce Au3+ to Au0 state forming gold nanoparticles and also to cap them (36). On the basis of this possibility we expect that the proteins extracted from the P. graveolens leaf reduce the Ag+ ions to Ag0 state and form silver nanoparticles. Presently it is not clear which are the compounds/proteins that are responsible for the reduction and capping of the silver nanoparticles. As mentioned previously, studies are in progress to identify these compounds/proteins, though it seems likely from the XRD and preliminary EDAX measurements that chlorophyll could be capping the silver nanoparticles.

Conclusion Figure 4. (a-d) TEM images at different magnifications of silver nanoparticles formed using P. graveolens leaf broth.

in the present case where proteins extracted from the P. graveolens leaf cap the silver nanoparticles, thereby stabilizing them. We caution that plants secrete a number of secondary metabolites covering a range of organic structures such as the terpenoids briefly discussed above. While the FTIR results indicate the presence of proteins and terpenoids in the geranium leaf broth, it is certainly possible that a number of other bioorganic compounds can exist in solution and participate in the reduction of silver ions and in the stabilization of the nanoparticles thus formed by surface capping. Efforts are currently underway to isolate the different bioorganic fractions in the geranium leaf broth and test them individually for silver ion reduction and binding with the nanoparticles. Figure 4 shows representative TEM pictures recorded from drop-coated films of the silver nanoparticles synthesized by treating silver nitrate solution with P. graveolens leaf broth for 24 h. At low magnification (Figure 4a), a very large density of silver nanoparticles can be seen. Thus, the silver nanoparticles are quite polydisperse and ranged in size from 16 to 40 nm with an average size ca. 27 nm. At higher magnification, the morphology of the silver nanoparticles is more clearly seen (Figure 4b and c). The particles are predominantly spherical with a small percentage being elongated (ellipsoidal). What is striking is that the nanoparticles appear to have assembled into very open, quasilinear superstructures rather a dense, closely packed assembly as is normally the case in aqueous nanoparticle solutions. The assembly of the silver nanoparticles into stringlike structures thus explains the appearance of a longitudinal component in the UV-vis spectra recorded from the silver nanoparticle solutions as discussed earlier (Figure 1). The assembly of the silver particles in the manner

The rapid synthesis of stable silver nanoparticles in high concentration using proteins/enzymes extracted from P. graveolens leaf was demonstrated. The reduction of the metal ions and stabilization of the silver nanoparticles is believed to occur by an enzymatic process. From the point of view of nanotechnology, this is a significant advance in being able to synthesize silver nanoparticles very rapidly compared to the previous reports. Achievement of such rapid time scales for synthesis of silver nanoparticles makes it more efficient as a biosynthetic pathway, though there still remains some scope for further decreasing the reduction time periods to make it a viable alternative to chemical synthesis methods.

Acknowledgment S.S.S. thanks the Council of Scientific and Industrial Research (CSIR), Govt. of India for a research fellowship. The TEM assistance of Ms. Renu Pasricha, Physical Chemistry Division, NCL Pune is gratefully acknowledged.

References and Notes (1) Simkiss, K.; Wilbur, K. M. Biomineralization; Academic Press: New York, 1989. (2) Biomimetic Materials Chemistry; Mann, S., Ed.; VCH Publishers: New York, 1996. (3) Lovley, D. R.; Stolz, J. F.; Nord, G. L.; Phillips, E. J. P. Anaerobic production of magnetite by a dissimilatory ironreducing microorganism. Nature 1987, 330, 252-254. (4) Philse, A. P.; Maas, D. Magnetic colloids from magnetotactic bacteria: chain formation and colloidal stability. Langmuir 2002, 18, 9977-9984. (5) Dickson, D. P. E. Nanostructured magnetism in living systems. J. Magn. Magn. Mater. 1999, 203, 46-49. (6) Mann, S. Molecular tectonics in biomineralization and biomimetic materials chemistry. Nature 1993, 365, 499-505. (7) Oliver, S.; Kupermann, A.; Coombs, N.; Lough, A.; Ozin, G. A. Lamellar aluminophopshates with surface patterns that

Biotechnol. Prog., 2003, Vol. 19, No. 6 mimic diatom and radiolarian microskeletons. Nature 1995, 378, 47-50. (8) Kroger, N.; Deutzmann, R.; Sumper, M.; Polycationic peptides from diatom biosilica that direct silica nanosphere formation. Science 1999, 286, 1129-1132. (9) Pum, D.; Sleytr, U. B. The application of bacterial S-layers in molecular nanotechnology. Trends Biotechnol. 1999, 17, 8-12. (10) Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Crystalline bacterial cell surface layers (S-layers): From supramolecular cell structure to biomimetics and nanotechnology. Angew. Chem., Int. Ed. 1999, 38, 1034-1054. (11) Stephen, J. R.; Maenaughton, S. J. Developments in terrestrial bacterial remediation of metals. Curr. Opin. Biotechnol. 1999, 10, 230-233. (12) Mehra, R. K.; Winge, D. R. Metal ion resistance in fungi. Molecular mechanisms and their regulated expression. J. Cell. Biochem. 1991, 45, 30-40. (13) Southam, G.; Beveridge, T. J. The occurrence of sulfur and phosphorus within bacterially derived crystalline and pseudocrystalline octahedral gold formed in vitro. Geochim. Cosmochim. Acta 1996, 60, 4369-4376. (14) Beveridge, T. J.; Murray, R. G. E. Sites of metal deposition in the cell wall of Bacillus subtilis. J. Bacteriol. 1980, 141, 876-887. (15) Fortin, D.; Beveridge, T. J. In Biomineralization. From Biology to Biotechnology and Medical Applications; Baeuerien, E., Ed.; Wiley-VCH: Weinheim, 2000. (16) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Silverbased crystalline nanoparticles, microbially fabricated. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13611-13614. (17) Klaus-Joerger, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Bacteria as workers in the living factory: metal-accumulating bacteria and their potential for materials science. Trends Biotechnol. 2001, 19, 15-20. (18) Joerger, R.; Klaus, T.; Granqvist, C. G. Biologically produced silver-carbon composite materials for optically functional thin-film coatings. Adv. Mater. 2000, 12, 407-409. (19) Nair, B.; Pradeep, T. Coalescence of nanoclusters and formation of submicron crystallites assisted by Lactobacillus strains. Cryst. Growth Des. 2002, 2, 293-298. (20) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Ramani, R.; Parischa, R.; Ajaykumar, P. V.; Alam, M.; Sastry, M.; Kumar, R. Bioreduction of AuCl4- ions by the fungus, Verticillium sp. and surface trapping of the gold nanoparticles. Angew. Chem., Int. Ed. 2001, 40, 3585-3588. (21) Mukherjee, P.; Senapati, S.; Mandal, D.; Ahmad, A.; Khan, M. I.; Kumar, R.; Sastry, M. Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum. ChemBioChem 2002, 3, 461-463. (22) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parischa, R.; Ajayakumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Fungus-mediated synthesis of silver nanoparticles and their immobilization in the mycelial matrix: A novel biological approach to nanoparticle synthesis. Nano Lett. 2001, 1, 515-519. (23) Ahmad, A.; Mukherjee, P.; Senapati, S.; Mandal, D.; Khan, M. I.; Kumar, R.; Sastry, M. Extracellular biosynthesis of

1631 silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf., B 2003, 28, 313-318. (24) Ahmad, A.; Mukherjee, P.; Mandal, D.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. Enzyme mediated extracellular synthesis of CdS nanoparticles by the fungus, Fusarium oxysporum. J. Am. Chem. Soc. 2002, 124, 12108-12109. (25) Ahmad, A.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. Extracellular biosynthesis of monodisperse gold nanoparticles by a novel extremophilic actinomycete, Thermomonospora sp. Langmuir 2003, 19, 3550-3553. (26) Gardea-Torresdey, J. L.; Parsons, J. G.; Gomez, E.; PeraltaVidea, J.; Troiani, H. E.; Santiago, P.; Jose-Yacaman, M. Formation and growth of gold nanoparticles inside live Alfalfa plants. Nano Lett. 2002, 2, 397-401. (27) Gardea-Torresdey, J. L.: Gomez, E.; Peralta-Videa, J. R.; Parsons, J. G.; Troiani, H.; Jose-Yacaman, M. Alfalfa sprouts: A natural source for the synthesis of silver nanoparticles. Langmuir 2003, 19, 1357-1361. (28) Gardea-Torresdey, J. L.; Tiemann, K. J.; Armendariz, V.; Bess-Oberto, L.; Chianelli, R. R.; Rios, J.; Parsons, J. G.; Gamez, G. Characterization of Cr(VI) binding and reduction to Cr(III) by the agricultural byproducts of Avena monida (Oat) biomass. J. Hazard. Mater. 2000, 80, 175-188. (29) Mulvaney, P. Surface plasmon spectroscopy of nanosized metal particles. Langmuir 1996, 12, 788-800. (30) Yong, P.; Rowson, N. A.; Farr, J. P. G.; Harris, I. R.; Macaskie, L. R. Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307. Biotechnol. Bioeng. 2002, 80, 369-379. (31) Cai, S.; Singh, B. R. Identification of β-turn and random coil amide III infrared bands for secondary structure estimation of proteins. Biophy. Chem. 1999, 80, 7-20. (32) Kota, Z.; Horvath, L. T.; Droppa, M.; Horrath, G.; Farkes, T.; Pali, T. Protein assembly and heat stability in developing thylakoid membranes during greening. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12149-12154. (33) Tavitian, B. A.; Nabedryk, E.; Mantele, W.; Breton, J. Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of primary reactions in photosystem I and photosystem II. FEBS Lett. 1986, 201, 151-157. (34) Rao, B. R. R.; Kaul, P. N.; Syamasundar, K. V.; Ramesh, S. Water soluble fractions of rose-scented geranium (Pelargonium species) essential oil. Bioresour. Technol. 2002, 84, 243-246. (35) Gole, A.; Dash, C. V.; Ramachandran, V.; Mandale, A. B.; Sainkar, S. R.; Rao, M.; Sastry, M. Pepsin-gold colloid conjugates: preparation characterization and enzymatic activity. Langmuir 2001, 17, 1674-1679. (36) Zhou, Y.; Chen, W.; Itoh, H.; Naka, K.; Ni, Q.; Yamane, H.; Chujo, Y. Preparation of a novel core-shell nanostructured gold colloid-silk fibroin bioconjugate by the protein in situ redox technique at room temperature. Chem. Commun. 2001, 2518-2519.

Accepted for publication May 30, 2003. BP034070W