Biohydrogen Production from Sucrose Using the Visible Light

important in environmental and energy development research (1-5). Some renewable biomass resources are starch, cellulose, sucrose, and lactose. These ...
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Bioconjugate Chem. 2003, 14, 268−272

Biohydrogen Production from Sucrose Using the Visible Light Sensitization of Artificial Zn Chlorophyll-a Yumi Takeuchi and Yutaka Amao* Department of Applied Chemistry, Oita University, Dannoharu 700, Oita 870-1192, Japan. Received July 24, 2002; Revised Manuscript Received November 1, 2002

A photoinduced hydrogen production system that couples sucrose degradation with invertase and glucose dehydrogenase (GDH) and hydrogen production with colloidal platinum as a catalyst using visible light-induced photosensitization of artificial Zn chlorophyll-a (Zn Chl-a) has been developed. Continuous hydrogen gas production over more than 240 min was observed when the reaction mixture containing sucrose, invertase, GDH, nicotinamide adenine dinucreotide (NAD+), Zn Chl-a, methyl viologen (MV2+, an electron relay reagent), and colloidal platinum was irradiated by visible light. Zn Chl-a was superior to that of Mg Chl-a in photostability and photosensitization activity.

INTRODUCTION

Hydrogen production systems from bioresources is important in environmental and energy development research (1-5). Some renewable biomass resources are starch, cellulose, sucrose, and lactose. These polysaccharides are hydrolyzed to form monosaccharides such as glucose. Thus, the conversion of glucose to hydrogen would be a useful new enzymatic pathway. Some studies on hydrogen production from glucose using an enzymatic pathway have been reported (6-10). Hydrogen production from glucose utilizing a combination of glucose dehydrogenase (GDH) and hydrogenase has been reported (11, 12). However, enzymatic photoinduced hydrogen production from other monosaccharides oligosaccharides or polysaccharides has received little attention. Glucose was obtained from sucrose by using invertase enzymatically. Thus, hydrogen production from oligosaccharides, such as sucrose, can be attained using a combination of invertase, GDH, and hydrogenase or some other hydrogen-evolving catalyst. On the other hand, some photoinduced hydrogen production systems consist of electron donor, photosensitizer, electron carrier, and catalyst. (13-19). For hydrogen-evolving catalyst, colloidal platinum (17-19) and hydrogenase from Desulfovibrio vulgaris (Miyazaki) (1316) are widely used in hydrogen production systems. Especially, colloidal platinum is stable against long-term irradiation. In photoinduced hydrogen production systems with visible light, as water-soluble zinc porphyrins have an absorption band in the visible light region (380600 nm), these porphyrins have been widely used as an effective photosensitizer (13, 14). Especially, zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) is useful as a photosensitizer (13, 14). However, the molar absorption coefficient of zinc porphyrins in the visible light region (500-600 nm) was lower than that in the near ultravisible light region (380-400 nm). On the other hand, Mg chlorophyll-a (Mg Chl-a), which acts as the effective photosensitizer in the photosynthesis of green plants (20), has an absorption maximum at 670 nm. Thus, Mg Chl-a * To whom correspondence should be addressed.Phone: +8197-554-7972. Fax: +81-97-554-7972. E-mail: [email protected].

is attractive as a visible photosensitizer. So far, photoinduced hydrogen production systems with chemically modified chlorophyll and hydrogenase (21, 22), and chlorophyll derivatives (23-25), were reported previously. However, Mg Chl-a purified from green plants is unstable to irradiation. On the other hand, zinc bacteriochlorophyll a was found in an aerobic bacterium, Acidiphilium rubrum (26). As zinc porphyrins are stable to irradiation and effective photosensitizers, zinc chlorophylls are attractive as stable visible photosensitizers. Some studies on the preparation and characterization of the zinc chlorophyll and bacteriochlorophylls have been reported (20, 27). In photoinduced hydrogen production with systems consisting of an electron donor, photosensitizer, electron relay, and catalyst, photoexcited photosensitizer reacts with the electron relay to form the reduced electron relay, hydrogen evolves by the proton reduction with the catalyst, and then the oxidized photosensitizer is reduced by the electron-donating reagent such as reduced nicotinamide adenine dinucreotide (NADH). Thus, the electron donor, NADH is the sacrificial reagent, and the oxidized electron donor, NAD+ is consumed in the reaction system. If NADH is regenerated, the photoinduced hydrogen production system is accomplished without NAD+ consumption. As GDH uses NAD+ as a cofactor, photoinduced hydrogen production with GDH, electron donor, photosensitizer, electron relay reagent, and catalyst can be attained. We previously reported visible lightinduced hydrogen production from sucrose using the photosensitization of Mg Chl-a (28). Thus, an effective photoinduced hydrogen production can be accomplished using Zn Chl-a as photosensitizer instead of Mg Chl-a. In this work, we describe the visible light-induced hydrogen production system coupling sucrose degradation with invertase and GDH, and hydrogen production with colloidal platinum using the photosensitization of artificial Zn Chl-a in the presence of methyl viologen (MV2+) as an electron relay reagent as shown in Scheme 1. EXPERIMENTAL PROCEDURES

General. Mg Chl-a from spirulina, invertase from yeast, and GDH from Bacillus sp. were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

10.1021/bc0255844 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/18/2002

Bioconjugate Chem., Vol. 14, No. 1, 2003 269 Scheme 1. Visible Light-Induced Hydrogen Production System Coupling the Sucrose Degradation with Invertase and GDH and Hydrogen Production with Colloidal Platinum Using the Photosensitization of Zn Chl-a in the Presence of MV2+

NAD+ and NADH were purchased from Oriental Yeast Co. Ltd. MV2+ dichloride and cetyltrimethylammonium bromide (CTAB) were purchased from Tokyo Kasei Co. Lid (Tokyo, Japan). Hydrogen hexachloplatinate hexahydrate and sodium citrate dihydrate were obtained from Kanto Chemical Co. Ltd (Tokyo, Japan). The other chemicals were analytical grade or the highest grade available. One unit of GDH activity was defined as the amount of enzyme that reduced 1.0 µmol of NAD+ to NADH by glucose per minute. One unit of invertase activity was defined as the amount of enzyme that produced 1.0 µmol of glucose by sucrose per minute. Preparation of Zn Chl-a. Zn Chl-a was synthesized by refluxing Mg Chl-a (50 mg, 56 µmol) with about 10 mol equiv of zinc acetate in 100 mL of methanol at 80 °C for 5 h. The insertion of zinc ion into the porphyrin ring of Chl-a was monitored by visible absorption spectroscopy. During the reaction, the characteristic absorption bands of Zn Chl-a at 421 and 662 nm increased and the absorbance bands at 433 and 668 nm of Mg Chl-a decreased gradually. After the mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and then the reaction mixture was washed with water to remove the unreacted zinc acetate. Finally, Zn Chl-a was precipitated in water. Zn Chl-a was collected by filtration and washed with water. The purification was performed by recrystallization (watermethanol). Zn Chl-a and Mg Chl-a were solubilized with 10 mmol dm-3 of CTAB, since Zn Chl-a and Mg Chl-a are insoluble in aqueous solution. Preparation of Colloidal Platinum. Colloidal platinum was prepared by reduction of hexachloplatinate solution with sodium citrate. The reduction procedure was similar to the previously reported method (17). A solution of 400 mL of water containing 30 mg of hydrogen hexachloplatinate hexahydrate was refluxed using a mantle heater and magnetic stirrer for 1.5 h, and then a solution of 30 mL of water containing 600 mg of sodium citrate dihydrate was added and refluxed with a magnetic stirrer at 100 °C for 4 h. The particle size of colloidal platinum prepared was estimated to be 1.5 nm. In general, the colloidal platinum activity decreased with increase the particle size (more than 2.0 nm) (17). The prepared colloidal platinum has the ability to release 0.7 µmol of hydrogen in the reaction system of 10 µL of colloidal platinum, 1.2 × 10-5 mmol of methyl viologen, and 7.7 × 10-5 mmol of sodium dithionite in 4.0 mL of 50 mmol dm-3 Tris-HCl buffer (pH 7.4) at 30 °C for 10 min. One unit of colloidal platinum activity was defined as the release of 1.0 µmol of hydrogen per minute. Spectroscopic Measurements. The UV-vis absorption spectrum of Zn Chl-a was recorded using a spectrophotometer (Multispec-1500 Shimadzu). The molar coefficients at the absorption maxima of Zn Chl-a were determined by the linear plot of absorbance versus Zn

Figure 1. UV-vis absorption spectra of Zn Chl-a (solid line) and Mg Chl-a (dashed line) in methanol solution. The concentrations of Zn Chl-a and Mg Chl-a were 79 and 16 µmol dm-3, respectively.

Chl-a concentration. The fluorescence emission spectrum of Zn Chl-a was measured using a spectrofluorophotometer with a 150 W xenon lamp as a visible excitation light source (RF-5300PC Shimadzu). The excitation and emission band-passes were 5.0 nm, respectively. The excitation wavelength was 600 nm. In these experiments, the absorbance at the excitation wavelength was kept constant at 0.2 for all the sample solutions. NADH Formation with Sucrose, Invertase, and GDH. The reaction was started by addition of NAD+ (0.80 µmol) solution to the sample solution containing sucrose (16 µmol), invertase (4.0 units), and GDH (5.0 units) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH 7.0). The reduction of NAD+ to NADH by GDH was monitored using a UV-vis spectrophotometer at 340 nm, with a molar extinction coefficient of 6.3 × 103 mol dm3 cm-1. Photoreduction of MV2+. Photoreduction of MV2+ was tested in the reaction mixture containing NAD+, sucrose, Zn Chl-a, MV2+, invertase, and GDH. The reaction system consisted of NAD+ (15 µmol), sucrose (0.30 mmol), Zn Chl-a (4.5 nmol), MV2+ (1.2 µmol), invertase (4.0 units), and GDH (5.0 units) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH 7.0). The sample solution was deaerated by repeated freeze-pump-thaw cycles and irradiated with a 200 W tungsten lamp at a distance of 3.0 cm, with a light intensity of 200 J m-2 s-1, at 30 °C. The light of the wavelength less than 390 nm was removed by a Toshiba L-39 cutoff filter (Tokyo, Japan). The reduction of MV2+ was monitored using a UV-vis spectrophotometer at 605 nm, with the molar extinction coefficient of 1.3 × 104 mol dm3 cm-1 (29). Visible Light-Induced Hydrogen Production. The photoinduced hydrogen production from sucrose was carried out as follows. The sample solution containing NAD+ (15 µmol), sucrose (0.30 mmol), Zn Chl-a (4.5 nmol), MV2+ (1.2 µmol), colloidal platinum (0.5 units), invertase (4.0 units), and GDH (5.0 units) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH 7.0) was deaerated by freeze-pump-thaw cycle six times, and substituted by argon gas. The amount of hydrogen evolved was measured by a Schimadzu GC-14B gas chromatograph (detector: TCD, column temperature: 40 °C, column: active charcoal with the particle size 60-80 mesh, carrier gas: nitrogen gas, carrier gas flow rate: 24 mL min-1). RESULTS AND DISCUSSION

UV-vis Absorption Spectrum of Zn Chl-a. Figure 1 shows the UV-vis absorption spectra of Zn Chl-a and Mg Chl-a in methanol solution. The absorption bands of

270 Bioconjugate Chem., Vol. 14, No. 1, 2003

Figure 2. Fluorescence emission spectra of Zn Chl-a (solid line) and Mg Chl-a (dashed line) in methanol solution. The excitation wavelength was 600 nm. The concentrations of Zn Chl-a and Mg Chl-a were 0.6 and 0.4 mmol dm-3, respectively.

Zn Chl-a were 421 nm attributed to Soret band and 662 nm attributed to Q-band. On the other hand, the absorption bands of Mg Chl-a were 433 nm attributed to Soret band and 668 nm attributed to Q-band. In comparison of the absorption spectra of Zn Chl-a and Mg Chl-a, a blue-shift in the absorption bands of Zn Chl-a was observed. Fluorescence Emission Spectrum of Zn Chl-a. Figure 2 shows fluorescence emission spectra of Zn Chl-a and Mg Chl-a in methanol solution. The fluorescence emission peak of Zn Chl-a and Mg Chl-a were 664 and 678 nm, respectively. In comparison of the fluorescence emission spectra of Zn Chl-a and Mg Chl-a, the blue-shift in the emission peak of Zn Chl-a was observed as well as that in the absorption spectra. The energies of the first excited singlet states of Zn Chl-a and Mg Chl-a were calculated from the average value of the frequencies of the longest wavelength of absorption maxima and the shortest wavelength of the fluorescence emission maxima. The energies of the first excited singlet states of Zn Chl-a and Mg Chl-a were 1.50 and 1.47 eV, respectively. NADH Formation with Sucrose, Invertase, and GDH System. When the sample solution containing sucrose, invertase, GDH, and NAD+ was incubated, the initial rate of NADH formation, which was determined by the amount of NADH with incubation for 15 min, was estimated to be (3.4 ( 0.1) × 10-8 mol min-1. After 80 min incubation, 0.77 ( 0.03 µmol of NADH was formed. The yield of conversion of NAD+ to NADH in this system was ca. 100%. When the sample solution containing glucose (16 µmol), GDH, and NAD+ (without sucrose and invertase) was incubated, the initial rate of NADH formation was estimated to be (4.2 ( 0.1) × 10-8 mol min-1. After 80 min incubation, 0.77 ( 0.02 of µmol NADH was formed, and the yield of conversion of NAD+ to NADH in this system also was ca. 100%. These results show that glucose formation from sucrose (process 1 in Scheme 1) with invertase proceeded rapidly. Stability of Zn Chl-a against Irradiation. The photostability of Zn Chl-a in the reaction mixture was investigated. The photostability was tested by irradiation with visible light using a 200 W tungsten lamp (light intensity of 200 J m-2 s-1). Figure 3 shows the absorbance changes at 662 nm attributed to the absorption maximum of Zn Chl-a (A) and at 670 nm attributed to the absorption maximum of Mg Chl-a (B) with irradiation time. The absorbance decrease of Zn Chl-a was slower than that of Mg Chl-a in solution as shown in curves A and B, indicating that the degradation rate of Zn Chl-a was suppressed compared with that of Mg Chl-a against

Figure 3. Photostability of Zn Chl-a solution under anaerobic conditions. Curves A and B are CTAB solubilized Zn Chl-a and Mg Chl-a aqueous solution, respectively. Samples were irradiated with visible light using 200 W tungsten lamp at a distance of 3.0 cm (light intensity of 200 J m-2 s-1).

Figure 4. The time dependence of the MV•+ concentration under steady-state irradiation with visible light using 200 W tungsten lamp at a distance of 3.0 cm (light intensity of 200 J m-2 s-1). (Closed circle): The sample solution consisting of invertase (4.0 units), GDH (5.0 units), NAD+ (15 µmol), sucrose (0.30 mmol), Zn Chl-a (4.5 nmol), and MV2+ (1.2 µmol) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). (Open circle): Mg Chl-a (3.0 nmol) was used as photosensitizer instead of Zn Chl-a. (Closed square): In the absence of NAD+.

irradiation. Thus, Zn Chl-a was superior to Mg Chl-a in photostability. Photoreduction of MV2+. Figure 4 shows the time dependence of the MV•+ concentration in the system containing NAD+, sucrose, Zn Chl-a, MV2+, invertase, and GDH with visible light irradiation (closed circle). The absorbance at 605 nm, absorption band of MV•+, increased with irradiation time. After 80 min irradiation, 0.69 µmol of reduced MV2+ was produced, and the yield of MV2+ to MV•+ was estimated to be ca. 58%. The photoreduction rate was independent of the concentrations of NAD+, sucrose, invertase, and GDH. On the other hand, the reduction rate depends on the concentrations of Zn Chl-a and MV2+. Thus, the rate-limiting step in the MV2+ reduction (processes 1, 2, and 3 in Scheme 1) is the photoinduced electron-transfer process from the photoexcited Zn Chl-a (*Zn Chl-a) to MV2+ (process 3 in Scheme 1). For the system using Mg Chl-a, 0.21 µmol of reduced MV2+ was produced, and the yield of MV2+ to MV•+ was estimated to be ca. 33% after 80 min irradiation (open circle). Thus, the photosensitization activity of Zn Chl-a was superior to that of Mg Chl-a. On the other hand, MV2+ was not reduced without NAD+ in the above system (closed square). There is no direct electron transfer between sucrose or glucose formed with invertase and MV2+, and between Zn Chl-a and MV2+. Thus, the visible light-induced MV2+ reduction proceeded by

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Figure 5. Time dependence of hydrogen production under steady-state irradiation with visible light using 200 W tungsten lamp at a distance of 3.0 cm (light intensity of 200 J m-2 s-1). (Closed circle): The sample solution consisting of invertase (4.0 units), GDH (5.0 units) NAD+ (15 µmol), sucrose (0.30 mmol), Zn Chl-a (4.5 nmol), MV2+ (1.2 µmol), and colloidal platinum (0.5 unit) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). (Open circle): Mg Chl-a (3.0 nmol) was used as photosensitizer instead of Zn Chl-a. (Closed square): In the absence of NAD+.

coupling the sucrose degradation with invertase and GDH (processes 1 and 2 in Scheme 1) and MV2+ reduction using the photosensitization of Zn Chl-a (process 3 in Scheme 1). Visible Light-Induced Hydrogen Production. As the MV2+ photoreduction system containing NAD+, sucrose, Zn Chl-a, MV2+, invertase and GDH was achieved, the photoinduced hydrogen production system was investigated. Figure 5 shows the time dependence of the photoinduced hydrogen production in the system containing NAD+, sucrose, Zn Chl-a, MV2+, colloidal platinum, invertase, and GDH by the visible light. By irradiation, hydrogen evolved continuously over more than 240 min. The amount of hydrogen production was estimated to be 7.0 µmol after 240 min irradiation. It indicated that 14 µmol of proton, that was 3111 times the amount of Zn Chl-a (4.5 nmol) in the sample solution, was reduced to hydrogen molecules. Therefore, the Zn Chl-a appeared to serve as the system for transferring electrons from NADH, which was formed from sucrose, to a more reductive hydrogen molecule. On the other hand, the hydrogen also was not evolved in the absence of NAD+ in the above system (closed square). There is no direct electron transfer between sucrose or glucose formed with invertase and colloidal platinum, and among Zn Chl-a, MV2+ and colloidal platinum. These results suggest that visible light-induced hydrogen production proceeded by coupling the sucrose degradation with invertase and GDH (processes 1 and 2 in Scheme 1) and the hydrogen production with colloidal platinum using the photosensitization of Zn Chl-a (processes 3 and 4 in Scheme 1). For the system using Mg Chl-a, the amount of hydrogen production was estimated to be 3.0 µmol after 240 min irradiation. It indicated that 6.0 µmol of proton, that was 2000 times the amount of Mg Chl-a (3.0 nmol) in the sample solution, was reduced to hydrogen molecules. Thus, the Zn Chl-a has a high activity of photosensitization compared with that of Mg Chl-a in the photoinduced hydrogen production system. Next let us focus on the relationship between the photoreduction of MV2+ and the photoinduced hydrogen production using Zn Chl-a and colloidal platinum. The formation of MV•+ reached a constant after 20 min irradiation as shown in Figure 4. This result suggests that the cycle of MV2+ and MV•+ was stable. As shown

in Figure 5, on the other hand, the photoinduced hydrogen production was proportional to irradiation time. As indicated from Figures 4 and 5, an efficient hydrogen or electron relay in the system may lead to efficient hydrogen production. In conclusion, a hydrogen production system that couples sucrose degradation with invertase and GDH and hydrogen production with colloidal platinum using visible light-induced photosensitization of artificial Zn Chl-a was developed, and continuous hydrogen gas was gained. Moreover, Zn Chl-a was superior to Mg Chl-a in photostability and photosensitization activity. Renewable biomass resources are effectively used to produce an environmentally clean energy source, hydrogen gas. However, since sucrose is expensive as a renewable biomass or clean energy source, hydrogen production from cellulose, which is an inexpensive renewable biomass, is desirable. Further research will be directed toward a photoinduced hydrogen production system that utilizes cellulose and cellulase instead of sucrose and invertase in process 1 of Scheme 1. ACKNOWLEDGMENT

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