Visible Light Induced Biohydrogen Production from Sucrose Using the

A photoinduced hydrogen production system, coupling sucrose degradation with invertase and glucose dehydrogenase (GDH) and hydrogen production with ...
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Bioconjugate Chem. 2002, 13, 898−901

Visible Light Induced Biohydrogen Production from Sucrose Using the Photosensitization of Mg Chlorophyll-a Yoshinobu Saiki and Yutaka Amao* Department of Applied Chemistry, Oita University, Dannoharu 700, Oita 870-1192, Japan. Received January 17, 2002; Revised Manuscript Received April 21, 2002

A photoinduced hydrogen production system, coupling sucrose degradation with invertase and glucose dehydrogenase (GDH) and hydrogen production with colloidal platinum as a catalyst using the visible light-induced photosensitization of Mg chlorophyll-a (Mg Chl-a), has been developed. Continuous hydrogen gas production was observed when the reaction mixture containing sucrose, invertase, GDH, nicotinamide adenine dinucleotide (NAD+), Mg Chl-a, methyl viologen (MV2+, an electron relay reagent), and colloidal platinum was irradiated by visible light.

INTRODUCTION

Hydrogen production from biomass resources is important in the environmental and alternative energy source fields (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 will be a useful new enzymatic pathway. Some studies on the hydrogen production from glucose using the enzymatic pathway have been reported (6-10). The hydrogen production from glucose with the combination of glucose dehydrogenase (GDH)1 and hydrogenase has been reported (11, 12). However, the enzymatic photoinduced hydrogen production from other monosaccharides, oligosaccharides, or polysaccharides has received little attention. Glucose was enzymatically obtained from sucrose by using invertase. Thus, hydrogen production from the oligosaccharide sucrose will be attained using the combination of invertase, GDH, and hydrogenase or some other hydrogen-producing catalyst. On the other hand, some photoinduced hydrogen production systems consist of electron donor, photosensitizer, electron carrier, and catalyst (13-19). Regarding hydrogen-producing catalysts, colloidal platinum (17-19) and hydrogenase from Desulfovibrio vulgaris (Miyazaki) (13-16) are widely used in hydrogen production systems. Especially, colloidal platinum is stable against long-term irradiation. In photoinduced hydrogen production systems with visible light, water-soluble zinc porphyrins have an absorption band in the visible light region (380600 nm), and these porphyrins have been widely used as effective photosensitizers (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 * To whom correspondence should be addressed. Phone: +81-97-554-7972. Fax: +81-97-554-7972. E-mail: amao@ cc.oita-u.ac.jp. 1Abbreviations: GDH, glucose dehydrogenase; NAD+, nicotinamide adenine dinucreotide; NADH, reduced nicotinamide adenine dinucreotide; MV2+, methyl viologen; MV•+, reduced methyl viologen; Mg Chl-a, Mg chlorophyll-a; ZnTPPS, zinc tetraphenylporphyrin tetrasulfonate; CTAB, cetyltrimethylammonium bromide.

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 Mg Chl-a in the Presence of MV2+

ultravisible light region (380-400 nm). On the other hand, Mg chlorophyll-a (Mg Chl-a), which acts as an effective photosensitizer in photosynthesis (20), has an absorption maximum at 670 nm. Thus, Mg Chl-a is attractive as a visible region photosensitizer. Photoinduced hydrogen production systems with chemically modified chlorophyll and hydrogenase (21, 22), and chlorophyll derivatives (23-25), were reported previously. In photoinduced hydrogen production with a system consisting of an electron donor, photosensitizer, electron relay, and catalyst, the photoexcited photosensitizer reacts with an electron relay to form a reduced electron relay, hydrogen evolves by proton reduction with the catalyst, and then the oxidized photosensitizer is reduced by an electron-donating reagent such as reduced nicotinamide adenine dinucreotide (NADH). Thus, the electron donor, NADH, was a sacrificial reagent, and the oxidized electron donor, NAD+, was 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 will be attained. In this work we describe the visible light-induced hydrogen production system that couples sucrose degradation with invertase and GDH, and hydrogen production with colloidal platinum using the photosensitization of Mg Chl-a in the presence of methyl viologen (MV2+) as an electron relay reagent as shown in Scheme 1.

10.1021/bc025506g CCC: $22.00 © 2002 American Chemical Society Published on Web 06/29/2002

Bioconjugate Chem., Vol. 13, No. 4, 2002 899 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). 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 hexachloroplatinate 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 NAD+ to NADH by glucose per minute. One unit of invertase activity was defined as the amount of enzyme that produced 1.0 µmol glucose by sucrose per minute. Mg Chl-a was solubilized with 10 mmol dm-3 of CTAB, since Mg Chl-a is insoluble in aqueous solution. Preparation of Colloidal Platinum. Colloidal platinum was prepared by reduction of hexachloroplatinate solution with sodium citrate. The reduction procedure was similar to the previous reported method (17). A solution of 400 mL of water containing 30 mg of hydrogen hexachloroplatinate hexahydrate was brought to boiling temperature using a mantle heater with a 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 a release of 1.0 µmol of hydrogen per minute. 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 UV-vis spectrophotometer at 340 nm, with the 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, Mg Chl-a, MV2+, invertase, and GDH. The reaction system consisted of NAD+ (15 µmol), sucrose (0.30 mmol), Mg Chl-a (3.0 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 freezepump-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 UV-vis spectrophotometer at 605 nm, with the molar extinction coefficient of 1.3 × 104 mol dm3 cm-1 (26). 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), Mg Chl-a (3.0

Figure 1. Time dependence of NADH formation with sucrose, invertase, NAD+, and GDH. (Closed circles): Sucrose (16 µmol), invertase (4.0 units), NAD+ (0.80 µmol), and GDH (5.0 units) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). (Open circles): Glucose (16 µmol), NAD+ (0.80 µmol), and GDH (5.0 units) in 3.0 mL of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). Each data point represents the mean of at least three separate experiments; bars are the standard error of the mean (SEM).

nmol), MV2+ (1.2 µmol), colloidal platinum (0.5 unit), invertase (4.0 units), and GDH (5.0 units) in 3.0 mol of 10 mmol dm-3 potassium phosphate buffer (pH 7.0) was deaerated by freeze pump thaw cycle for six times and substituted by argon gas. The amount of hydrogen evolved was measured by Schimadzu GC-14B gas chromatography (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

NADH Formation with Sucrose, Invertase, and GDH System. When the sample solution containing sucrose, invertase, GDH, and NAD+ was incubated, the of the NADH was formed in a time-dependent manner (Figure 1, closed circle). 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 the 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, NADH was formed in a time-dependent manner (Figure 1, open circle), and 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 µmol of NADH was formed, and the yield of the 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 Mg Chl-a against Irradiation. The photostability of Mg Chl-a in the reaction mixture was investigated. The photostability was tested by irradiation with visible light using 200 W tungsten lamp (light intensity of 200 J m-2 s-1). Figure 2 shows the absorbance change at 670 nm attributed to the absorption maximum of Mg Chl-a with irradiation time. CTAB solublized Mg Chl-a solution was rapidly bleached by irradiation (curve A). On the other hand, the absorbance decrease of Mg Chl-a in the presence of NAD+ and NADH was slower as shown in curve B and C, indicating that the degradation of Mg Chl-a was suppressed by the addition of NAD+

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Figure 2. Photostability of Mg Chl-a solution under anaerobic condition. Curve A, CTAB solubilized Mg Chl-a aqueous solution. Curve B and C, CTAB solubilized Mg Chl-a aqueous solution in the presence of NAD+ and NADH, 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 3. 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). The sample solution consisting of invertase (4.0 units), GDH (5.0 units), NAD+ (15 µmol), sucrose (0.30 mmol), Mg Chl-a (3.0 nmol), and MV2+ (1.2 µmol) in 3.0 mol of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). (Open circles): In the absence of NAD+. Each data point represents the mean of at least three separate experiments; bars are the SEM.

and NADH. The photoinduced degradation of Mg Chl-a proceeded by the reaction between photoexcited Mg Chl-a molecules. In the presence of the electron donor or acceptor reagent such as NAD+ or NADH, on the other hand, the electron transfer from the photoexcited Mg Chl-a to the electron acceptor reagent or from the electron donor reagent to the photoexcited Mg Chl-a proceeded. These electron transfer processes rapidly proceeded compared with that of the reaction between photoexcited Mg Chl-a molecules. Thus, the reaction between photoexcited Mg Chl-a molecules was suppressed by the electron donor or acceptor reagent such as NAD+ or NADH. Photoreduction of MV2+. Figure 3 shows the time dependence of the MV•+ concentration in the system containing NAD+, sucrose, Mg Chl-a, MV2+, invertase, and GDH with visible light irradiation. The absorbance at 605 nm, absorption band of MV•+, increased with irradiation time. After 80 min irradiation, (0.2 ( 0.01) µmol reduced MV2+ was produced and the yield of MV2+ to MV•+ was estimated to be ca. 33%. 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 Mg Chl-a

Figure 4. 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 circles): The sample solution consisting of invertase (4.0 units), GDH (5.0 units) NAD+ (15 µmol), sucrose (0.30 mmol), Mg Chl-a (3.0 nmol), MV2+ (1.2 µmol), and colloidal platinum (0.5 unit) in 3.0 mol of 10 mmol dm-3 potassium phosphate buffer (pH ) 7.0). (Open circles): In the absence of NAD+. Each data point represents the mean of at least three separate experiments; bars are the SEM.

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 Mg Chl-a (*Mg Chl-a) to MV2+ (process 3 in Scheme 1). On the other hand, MV2+ was not reduced without NAD+ in the above system (open circle). There is no direct electron transfer between sucrose or glucose formed with invertase and MV2+, and between Mg Chl-a and MV2+. Thus, the visible light-induced MV2+ reduction proceeded by coupling the sucrose degradation with invertase and GDH (processes 1 and 2 in Scheme 1) and MV2+ reduction using the photosensitization of Mg Chl-a (process 3 in Scheme 1). Visible Light-Induced Hydrogen Production. As the MV2+ photoreduction system containing NAD+, sucrose, Mg Chl-a, MV2+, invertase, and GDH was achieved, the photoinduced hydrogen production system was investigated. Figure 4 shows the time dependence of the photoinduced hydrogen production in the system containing NAD+, sucrose, Mg Chl-a, MV2+, colloidal platinum, invertase, and GDH by the visible light. By irradiation, hydrogen evolved continuously for more than 420 min. The amount of hydrogen production was estimated to be 4.3 ( 0.1 µmol after 420 min irradiation. It indicated that 8.6 µmol of proton, that was 2867 times the amount of Mg Chl-a (3.0 nmol) in the sample solution, was reduced to hydrogen molecules. Therefore, the Mg Chl-a appeared to serve as the system for transferring electrons from NADH, which was formed from sucrose conversion to gluconic acid, to a more reductive molecule. It should be noted that hydrogen was not evolved in the absence of NAD+ in the above system (open circles, Figure 4). There is no direct electron transfer between sucrose or glucose formed with invertase and colloidal platinum, and among Mg Chl-a, MV2+, and colloidal platinum. These results suggest that the visible lightinduced 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 Mg Chl-a (processes 3 and 4 in Scheme 1). In conclusion, a hydrogen production system coupling sucrose degradation with invertase and GDH, and hydrogen production with colloidal platinum using the

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visible light-induced photosensitization of Mg Chl-a, was developed, and continuous generation of hydrogen gas was achieved. Renewable biomass resources are effectively used to produce an environmentally clean energy source, hydrogen gas. In the present system, fructose consumption occurred; glucose and fructose were formed from sucrose by invertase. The system involving fructose dehydrogenation is being studied in detail. ACKNOWLEDGMENT

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