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Photobiological hydrogen production
JOURNAL OF BIOSCIENCE AND Vol. 88, No. 1, 1-6. 1999
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REVIEW Photobiological Hydrogen Production YASUO ASADA’* AND JUN MIYAKE2,’ National Institute of Bioscience and Human Technology, AIST/MITI, 1-I Higashi, Tsukuba-shi, Ibaraki 305-8566’ and National Institute for Advanced Interdisciplinary Research, AIST/MITI, l-l-4 Higashi, Tsukuba-shi, Ibaraki 305-8562,2 Japan Received 20 May 1999/Accepted 3 June 1999
The principles and recent progress in the research and development of photobiological hydrogen production are reviewed. Cyanobaeteria produce hydrogen gas using nitrogenase and/or hydrogenase. Hydrogen production mediated by native hydrogenases in cyanobacteria occurs under in the dark under anaerobic conditions by degradation of intracellular glycogen. In vitro and in vivo coupling of the cyanobacterial photosynthetic system with a clostridial hydrogenase via cyanobacterial ferredoxin was demonstrated in the presence of light. Genetic transformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum was successful: the active enzyme was expressed in PCC7942. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Coculture of Rhodobacter and CZostriudium was applied for hydrogen production from glucose. A mutant strain of Rhodobacter sphaeroides RV whose light-harvesting proteins were altered was obtained by UV irradiation. Hydrogen productivity by the mutant was improved when irradiated with monochromatic light of some wavelengths. The development of photobioreactors for hydrogen production is also reviewed. [Key words: hydrogen,
cyanobacteria,
photosynthetic
Our most important energy sources have been fossil fuels. The oil crisis of the 70’s demonstrated to us the fragility of energy supply structure in our modern civilization, and promoted research on new energy production methods, including photobiological hydrogen production. Recovery and stabilization of oil supply might have partially discouraged efforts on energy research. However, the recent worldwide ‘greenhouse effect’ attributed to an increasing concentration of carbon dioxide in the air due to combustion of fossil fuels has been again encouraged research for alternative energy sources to fossil fuels. The new technologies that are to mitigate these energy and environmental problems should therefore also be environmentally acceptable. Photobiological hydrogen production (l-3, for review) could be a potentially environmentally acceptable energy production method because hydrogen gas is renewable using the primary energy source, sunlight, and does not liberate carbon dioxide during combustion. The lower conversion efficiency of a biological system than to a solar battery could be compensated for by its lower requirement for energy and the lower initial investments. In this paper, we will review principles and recent progress in the development of photobiological hydrogen production methods, with reference to our works which were carried out in fiscal year 1991 to 1998 as a part of collaborative research with RITE (refer to Acknowledgement).
There are two kinds of production, hydrogenases types of photosynthesis, anoxygenic photosynthesis,
enzymes catalyzing hydrogen and nitrogenases, and two oxygenic photosynthesis and which can be carried out by
* Corresponding author. Present address: Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama-shi 701-1296, Japan.
bacteria, photobioreactor]
two groups of microorganisms, the cyanobacteria/green algae and the photosynthetic bacteria, respectively. We will review each combination of the enzyme and microorganism for hydrogen production. NITROGENASE-MEDIATED HYDROGEN PRODUCTION IN CYANOBACTERIA In 1974, Benemann and Weare (4) demonstrated that a nitrogen-fixing cyanobacterium, Anabaena cylindrica, produced hydrogen mediated by nitrogenase (5) and oxygen gas simultaneously in argon atmosphere for several hours. Nitrogenase mediates reduction of molecular nitrogen to ammonium with consumption of reducing power (mediated by ferredoxin) and ATP. However, nitrogenase can catalyze reduction of a proton in the absence of nitrogen gas (i.e., argon atmosphere). Furthermore, hydrogen production by nitrogenase enzyme occurs as a side reaction at the rate of one third to fourth of that of nitrogen fixation even in a 100% nitrogen gas atmosphere. The nitrogenase enzyme, itself, is extremely oxygenlabile. Cyanobacteria have a well-developed mechanism for the protection of nitrogenase from oxygen gas that can simultaneously supply both ATP and reducing power. The most successful strategy has been developed by a heterocystous and filamentous group of the organisms (6). Nitrogenase enzyme is localized in the heterocysts. Vegetative cells in filamentous cyanobacteria carry out oxygenic photosynthesis. Organic compounds produced by carbon dioxide reduction are transferred into heterocysts and decomposed to provide reducing power to nitrogenase. ATP can be provided by anoxygenic photosynthesis by Photosystem I in heterocysts. There have been several studies aimed at the prolongation and optimization of hydrogen production by nitrogen-fixing cyanobacteria (7-9).
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The physiological roles of ‘reversible’ hydrogenases in nitrogen-fixing cyanobacteria have been controversial, but ‘uptake’ hydrogenase is estimated to consume hydrogen gas for reutilization, which results in a decrease in the net hydrogen production (10). We reported aerobic hydrogen production (11) by a nitrogen-fixing Anabaena sp. (12) that was supposed to be an uptake hydrogenase-deficient strain. After 12d culture starting with a low inoculum concentration, the strain accumulated approximately 10% hydrogen and 70% oxygen gas in the gas phase of the vessel as products of the side reaction of nitrogenase. Recently, Hall and coworkers (13) also reported aerobic hydrogen production by using a Nostoc mutant that was deficient in ‘uptake’ hydrogenase. Miyamoto et al. (14) demonstrated outdoor hydrogen production by A. cylindrica in California. A nitrogenstarved culture of the cyanobacterium was continuously sparged by an argon-based gas mixture, and the hydrogen concentration in the effluent gas was measured. The average conversion efficiency over a one-month period (combustion energy of hydrogen gas produced by the cyanobacterium/incident solar energy in the photobioreactor area) was estimated to be approximately 0.2%. Mitsui and coworkers have extensively screened for cyanobacteria with high hydrogen productivity (15). Miami BG7, one of their most potent hydrogen producers was tested for outdoor culture (16). They isolated the unicellular and aerobic nitrogen-fixing Synechococcus sp. Miami BG0435 11, also and proposed a new strategy in unicellular and aerobic species to protect and drive oxygenlabile nitrogenase enzyme in oxygen-producing cells by developing synchronous culture techniques (17). This strain is a potent hydrogen producer. They estimated a conversion efficiency of 3.5% based on par (photosynthetically active irradiation that includes light energy of 400-700 mm in wavelength) using an artificial light source (18). HYDROGENASE-MEDIATED HYDROGEN PRODUCTION IN GREEN ALGAE AND CYANOBACTERIA Gaffron and Rubin (19) reported that a green alga, Scenedesmus, produces molecular hydrogen not only in the presence of light but also in the dark under anaerobic conditions. The enzyme mediating the hydrogen production was revealed to be hydrogenase (20, for review), catalyzing the reaction as follows: = HZ + 2Xoxidieed Z-I+ + &educed The electron carrier, X, is considered to be ferredoxin in green algae. Since ferredoxin is reduced with water during oxygenic photosynthesis, the green alga can be theoretically considered as being a water-splitting microorganism. In many cases, however, reducing power (electron donor) for hydrogenase is not always derived from the splitting of water but may partially from intracellular organic compounds such as starch in the presence of light. Green algae were considered as being light-dependent, water-splitting catalysts but hydrogen production using green algae was not feasible. Their hydrogenase enzyme is too oxygen-labile for sustainable hydrogen production: light-dependent hydrogen production ceases within minutes since the oxygen gas produced photosynthetically inhibits or inactivates hydroge-
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nase at less than 1% in the gas phase. A continuous gas flow system for maintaining a low oxygen concentration in a reaction vessel (21) seemed to be applicable for basic studies but for as a practical system. Seibert and coworkers (22) have been attempting to isolate mutants of Chlarnydomonas that posses an oxygen-tolerant hydrogenase. Green algae could be applied for another method of hydrogen production: use of dark and fermentative hydrogen production. Miura et al. (23-25) proposed hydrogen production in light/dark cycles. According to their proposal, carbon dioxide is reduced to starch by photosynthesis in the daytime and then the starch is decomposed to hydrogen gas, organic acids and alcohols under anaerobic conditions during the nighttime. We have reported that cyanobacteria can produce hydrogen in the dark under anaerobic conditions (2630). Spirulina species were demonstrated to have the highest hydrogen producing activities among those tested cm. The response by hydrogen-producing cyanobacterium, Mcrocystis aeruginosa to light is different to that of green algae (29). Light irradiation did not stimulate hydrogen production but resulted in hydrogen consumption. The light-dependent hydrogen uptake was not inhibited by the inhibitor of photosystem II, DCMU (3(3,Cdichloro-phenyl)-1,1-dimethyl urea), but inhibited by the antagonist of plastoquinone, DBMIB (2,5dibromo3-methyl-6-isopropyl-p-benzoquinone). These results suggest that hydrogen produced by degradation of endogenous glycogen may be taken up by the light-dependent reaction of photosystem I via plastoquinone. We partially purified a hydrogenase from A4. aeruginosa (31). The electron carrier for hydrogenase in cyanobacteria has still not been identified (30, 31). Recently, the genes cyanobacterial hydrogenases have cloned, and a new hypothesis relating to photosystem and the respiratory system was proposed (32). A NEW SYSTEM FOR HYDROGEN PRODUCTION: COMBINATION OF THE CYANOBACTERIAL PHOTOSYNTHETIC SYSTEM WITH CLOSTRIDIAL HYDROGENASE As described in the first section, some nitrogen-fixing cyanobacteria could be potential candidates for practical application to hydrogen production. Hydrogen production by nitrogenase, however, is an energy-consuming process due to hydrolysis of ATP molecules (at least 4 per 1 mol of hydrogen). On the other hand, hydrogenasemediated hydrogen production by cyanobacteria and green algae is economic due to the nonrequirement of ATP, although it is not sustainable under light conditions. The ideal and ultimate hydrogen producing system could be the sustainable hydrogenase-mediated, watersplitting system. Our strategy involves coupling of clostridial hydrogenase with the oxygenic photosynthetic system via ferredoxin (Fig. 1). To check the relevance of our strategy, in vivo coupling was tested; hydrogenase protein was electrointroduced into cyanobacterial cells (33). The cyanobacterial cells that contained clostridial hydrogenase and then treated with protease produced hydrogen gas upon light irradiation. Then, we attempted to overexpress the hydrogenase in a cyanobacterium, Synechococcus PCC7942, by developing a genetic engineering system (34-37). The adjustment
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FIG. 1. Schematic of photosynthetic (A) and transformant (B) cyanobacteria.
electron flows in wild-type
of the Shine-Dalgarno sequence to the host cyanobacteriurn was supposed to be critical for heterologous expression (38). Finally, we succeeded in heterologous expression of the hydrogenase (Asada et al., in submission) which was thought to be very difficult since the hydrogenase gene is composed of many accessory gene elements (10). We are presently characterizing the transformant strain, particularly in relation to its genetically introduced hydrogenase activity. HYDROGEN PRODUCTION BY PHOTOSYNTHETIC BACTERIA
Photosynthetic bacteria carry out anoxygenic photosynthesis using organic compounds and reduced sulfur compounds as electron donors, which are categorized as non-sulfur and sulfur photosynthetic bacteria, respectively. Some non-sulfur photosynthetic bacteria are potent hydrogen producers utilizing organic acids such as lactic, succinic and butyric acids or alcohols. Since light energy is not used for water oxidation, the efficiency of light energy for the production of hydrogen by photosynthetic bacteria is theoretically much higher than that by cyanobacteria. From the practical point of view, photosynthetic bacteria are important since they can be used for dual purposes of waste water treatment and hydrogen production (39, 40). Recycling of agricultural wastes and various biomasses has been also realized simultaneously with hydrogen production by photosynthetic bacteria. Ike et al. demonstrated that raw algal biomass can be used as a resource for hydrogen production. They proposed chemical digestion of the algal mass, to produce good substrates for photosynthetic bacteria from starch, a major constituent of algal biomass, to improve the yield of hydrogen (41). They also demonstrated fermentation of living algal biomass by a lactic acid bacterium (42-44). Lactic acid is an excellent substrate for hydrogen production by photosynthetic bacteria, and a maximum hydrogen yield of 8 mol/mol starch-glucose from algal biomass was observed. The enzyme catalyzing hydrogen production by photosynthetic bacteria is nitrogenase (5), whereas hydrogenases may be active for hydrogen uptake in many photosynthetic bacteria. Miyake and Kawamura (45) demonstrated that 6 to 8% of the incident light energy can be converted to hydrogen gas (combustion energy) in laboratory experiments using our isolate, Rhodobacter sp. 8703 (46) with lactic acid as an electron donor.
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Anaerobic bacteria degrade sugars to hydrogen gas and organic acids (47, i.e.), but cannot utilize the organic acids as electron donors. Miyake et al. (48) demonstrated the combined use of photosynthetic bacteria with anaerobic bacteria for complete conversion of sugar to hydrogen. Theoretically, 1 mol of glucose can be converted to 12mol of hydrogen, and their experimental result was 7 mol of hydrogen per mole of consumed glucose. Carbohydrate polymers such as starch or cellulose, the major constituents of agricultural wastes and biomasses, are rarely degraded by photosynthetic bacteria alone for hydrogen production. Ike et al. proposed a one-step hydrogen production from starch using the halophilic bacterial community originating from activated sludge (49, 50). In this community, starch could be converted to hydrogen directly by the cooperative action of Rhodobium marinum and Vibrio Jluvialis. From the practical point of view, organic wastes frequently contain sugar or sugar polymers. The combined use of the two kinds of bacteria would enhance their probability of application in photobiological hydrogen production (51). GENETIC ENGINEERING TO CONTROL EXPRESSION OF THE PHOTOSYNTHETIC PROTEINS
For efficient hydrogen production, the balance between a photosystem and hydrogen producing enzyme is required. For cyanobacteria, overexpression of heterologous hydrogenase was attempted as described above. Compared to nitrogenase activity in photosynthetic bacteria, too much light energy is converted to biochemical energy by the photosystem, which is lost via other biochemical processes. We attempted to reduce the photosystem content to an appropriate level for the present nitrogenase activity. We have developed a new genetic engineering method to control the expression of photosynthetic protein complexes; the promoter of the puf operon from Rhodobacter sphaeroides RV encoding the photoreaction center and light-harvesting proteins (52) was introduced into the host to absorb active ribosomes to prevent transcription of the puf operon. Furthermore, we have obtained by UV irradiation a mutant, P3, with altered composition of light-harvesting complexes (53). Hydrogen production by the P3 mutant was characterized by using a hydrogen electrode system under monochromatic light of several wavelengths. When 800 nm and 850nm monochromatic light was irradiated, the mutant strain produced hydrogen gas at a higher rate than the wild type (Fig. 2). These results suggest that the composition of lght-harvesting proteins may finally affect the productivity of hydrogen gas. RESEARCH AND PHOTOBIOREACTORS HYDROGEN
DEVELOPMENT OF FOR PHOTOBIOLOGICAL PRODUCTION
‘Self-shadowing’ has posed a difficult problem with regard to the utilization of solar energy by cultures of photosynthetic microorganisms. Quite a small portion of photobioreactors is actually active due to the inability of the light penetrate deeply when microbial cell concen-
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FIG. 2. Comparison of hydrogen production rates by the wild-type and P3 mutant strain of Rb. sphueroides RV (55). Symbols: 0, wild strain; 0, P3 mutant. (a) 800 nm; (b) 850 nm; (c) 875 nm. Refer (55) for detailed experimental conditions.
tration is high. Therefore, a basic analysis on the distribution of light intensity and shift in composition wavelength in relation to hydrogen production (55-57) is important. Wakayama et al. studied on effects of chopped light on hydrogen production by photosynthetic bacteria (58). Moreover, immobilization of bacteria could contribute to prolongation, stabilization and ease in exchange of culture broth. We have developed several immobilization methods using glass plates, glass beads and agar gels (59-62). A photobioreactor for hydrogen production by cyanobacteria was studied by Markov et al. (63). Various types of photobioreactors in RITE Hydrogen Project (refer to Acknowledgments) has been demonstrated (6467). The largest photobioreactor (Fig. 3) developed for hydrogen production is a floating type (4001 working volume) (67).
The future and technological applicability of biological hydrogen production depends not only on progress in research, i.e., improvement of efficiency by breeding or developing bioreactor, but also on economical factors such as of fossil fuels, and social acceptance and development of hydrogen energy systems. ACKNOWLEDGMENTS Our studies were partially performed as a part of R & D Projects of Environmentally-Friendly Technology for the Production of Hydrogen under the management of RITE (Research Institute of Innovative Technologies for the Earth) which was sponsored by MITI (Ministry of International Trade and Industry) with the supports of the New Energy and Industrial Technology Development Organization (NEDO). REFERENCES
FUTURE PROSPECTS We consider that biological hydrogen production is the most challenging undertaking issue in the field of biotechnology for overcoming environmental problems.
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M.: Biological production of hydrogen from cellulose by natural anaerobic microflora. J. Ferment. Bioeng., 79, 395-397 (1995). Miyake, J., Mao, X.-Y., and Kawamura, S.: Photoproduction of hydrogen from glucose by a co-culture of a photosynthetic bacterium and Clostridium butyricum. J. Ferment. Technol., 62, 531-535 (1984). Ike, A., Toda, N., Murakawa, T., Hirata, K., and Miyamoto, K.: Hydrogen photoproduction from starch in C02-fixing microalgal biomass by a halotorelant bacterial community, p. 311-318. In Zaborsky, 0. R. (ed.), BioHydrogen. Plenum Press, London (1998). Ike, A., Murakawa, T., Kawaguchi, H., Hirata, K., and Miyamoto, K.: Photo-production of hydrogen from raw starch using a halophilic bacterial community. J. Biosci. Bioeng., 88 (1999). (in press) Zhu, H.-G., Suzuki, T., Tsygankov, A. A., Nakada, E., Asada, Y., and Miyake, J.: Hydrogen production from waste water (alcohol and tofu waste water) by photosynthetic bacteria and Clostridium. J. Water Treatment, 10 (1995). Nagamine, Y., Kawasugi, T., Miyake, M., Asada, Y., and Mlyake, J.: Characterization of a photosynthetic bacterium Rhodobacter sphaeroides RV for hydrogen production. J. Marine Biotechnol., 4, 34-37 (1996). Vasilyeva, L., Miyake, M., Hara, M., Nakada, E., Nishikata, S., Asada, Y., and Miyake, J.: Characterization of a novel light-harvesting mutant of Rhodobacter sphaeroides with relation to photohydrogen, p. 123-131. In Zaborsky, 0. R. (ed.), BioHydrogen. Plenum Press, London (1998). Vasilyeva, L., Miyake, M., Khatipov, E., Wakayama, T., Sekine, M., Hara, M., Nakada, E., Asada, Y., and Miyake, J.: Enhanced hydrogen production by a mutant of Rhodobacter sphaeroides having an altered light-harvesting system. J. Biosci. Bioeng., 87 (1999). (in press) Nakada, E., Asada, Y., Arai, T., and Miyake, J.: Light penetration into cell suspension of photosynthetic bacteria and relation to hydrogen production. J. Ferment. Bioeng., 80, 5357 (1995). Nakada, E., Nishikata, S., Asada, Y., and Miyake, J.: Effect of culture depth and light wavelength for hydrogen production by photosynthetic bacteria, p. 837-840. In Mathis, P. (ed), Photosynthesis: from light to biosphere (Proc. 10th Int. Photosynthesis Congress, Montpellier, France, 20-25 Aug.), vol. 5. Kluwer Academic Publishers, Dordrecht (1995). Nakada, E., Nishikata, S., Asada, Y., and Miyake, J.: Light penetration and wavelength effect on photosynthetic bacteria culture for hydrogen production, p. 345-352. In Zaborsky, 0. R. (ed.), BioHydrogen. Plenum Press, London (1998).
J. BIOSCI. BIOENG.. 58. Wakayama, T., Toriyama, A., Kawasugi, T., Arai, T., Asada, Y., and Miyake, J.: Photohydrogen production using photosynthetic Rhodobacter sphaeroides RV: simulation of light cycles of natural sunlight using artificial source, p. 375-381. In Zaborsky, 0. R. (ed.), BioHydrogen. Plenum Press, London (1998). 59. Tsygankov, A. A., Hi&a, Y., Asada, Y., aud Miyake, J.: Immobilization of the purple non-sulfur bacterium Rhodobacter sphaeroides on glass surfaces. Biotechnol. Techniques, 7, 283286 (1993). 60. Tsygankov, A. A., Hi&a, Y., Miyake, M., Asada, Y., and Miyake, J.: Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction. J. Ferment. Bioeng., 7, 575-578 (1994). 61. Nakada, E., Nishikata, S., Asada, Y., and Miyake, J.: Hydrogen production by gel-immobilized cells of Rhodobacter sphaeroides: distribution of cells, pigments, and hydrogen evolution. J. Marine Biotechnol., 4, 38-42 (1996). 62. Zhu, H.-G., Suzuki, T., Tsygankov, A., Asada, Y., and Miyake, J.: Hydrogen production from tofu waste water by Rhodobacter sphaeroides immobilized in agar gels. Int. J. Hydrogen Energy, 24, 305-310 (1999). 63. Markov, S. A., Bazin, M. J., and Hall, D. 0.: The potential of using cyanobacteria in photobioreactors for hydrogen production. Advances in Biochem. Eng. Biotech., 52, 59-86 (1995). 64. D’Addario, E., Fascetti, E., and Valdiserri, M.: Hydrogen production from organic wastes by continuous culture of Rhodobacter sphaeroides, p. 2577-2582. In Veziroglu, T. N., Winter, C.-J., Baselt, J. P., and Kreysa, G. (ed.), Hydrogen energy progress XI, vol. 3. (Proc. 11th World Hydrogen Energy Conf., Stuttgart, Germany, 23-28 June) Dechema, Frankfurt (1996). 65. Miuami, M., Uechi, T., Kimura, M., Nishishiro, T., Kuriaki, H., Sano, K., and Kawasugi, T.: Hydrogen production by photosynthetic bacteria using sewage sludge, p. 2583-2588. In Veziroglu, T. N., Winter, C.-J., Baselt, J. P., and Kreysa, 0. (ed.), Hydrogen energy progress XI, vol. 3. (Proc. 11th World Hydrogen Energy Conf., Stuttgart, Germany, 23-28 June) Dechema, Frankfurt (1996). 66. Morimoto, M., Kawasaki, S., El-Shishtawy, R. M. A., Ueno, Y., and Kunishi, N.: Solar energy-induced and diffused photobioreactor. v. 2761-2766. In Veziroglu. T. N.. Winter. C.-J., Baselt, J. P.: grid Kreysa, G. (ed.), Hydrogen energy progress XI, vol. 3. (Proc. 11th World Hydrogen Energy Conf., Stuttgart, Germany, 23-28 June) Dechema, Frankfurt (1996). 67. Ohtsuki, T., Uchiyama, S., Fujiki, K., and Fukunaga, S.: Hydrogen production by a floating-type photobioreactor, p. 369-374. In Zaborsky, 0. R. (ed.), BioHydrogen. Plenum Press, London (1998).
BIOENGINEERING
REVIEW Photobiological Hydrogen Production YASUO ASADA’* AND JUN MIYAKE2,’ National Institute of Bioscience and Human Technology, AIST/MITI, 1-I Higashi, Tsukuba-shi, Ibaraki 305-8566’ and National Institute for Advanced Interdisciplinary Research, AIST/MITI, l-l-4 Higashi, Tsukuba-shi, Ibaraki 305-8562,2 Japan Received 20 May 1999/Accepted 3 June 1999
The principles and recent progress in the research and development of photobiological hydrogen production are reviewed. Cyanobaeteria produce hydrogen gas using nitrogenase and/or hydrogenase. Hydrogen production mediated by native hydrogenases in cyanobacteria occurs under in the dark under anaerobic conditions by degradation of intracellular glycogen. In vitro and in vivo coupling of the cyanobacterial photosynthetic system with a clostridial hydrogenase via cyanobacterial ferredoxin was demonstrated in the presence of light. Genetic transformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum was successful: the active enzyme was expressed in PCC7942. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Coculture of Rhodobacter and CZostriudium was applied for hydrogen production from glucose. A mutant strain of Rhodobacter sphaeroides RV whose light-harvesting proteins were altered was obtained by UV irradiation. Hydrogen productivity by the mutant was improved when irradiated with monochromatic light of some wavelengths. The development of photobioreactors for hydrogen production is also reviewed. [Key words: hydrogen,
cyanobacteria,
photosynthetic
Our most important energy sources have been fossil fuels. The oil crisis of the 70’s demonstrated to us the fragility of energy supply structure in our modern civilization, and promoted research on new energy production methods, including photobiological hydrogen production. Recovery and stabilization of oil supply might have partially discouraged efforts on energy research. However, the recent worldwide ‘greenhouse effect’ attributed to an increasing concentration of carbon dioxide in the air due to combustion of fossil fuels has been again encouraged research for alternative energy sources to fossil fuels. The new technologies that are to mitigate these energy and environmental problems should therefore also be environmentally acceptable. Photobiological hydrogen production (l-3, for review) could be a potentially environmentally acceptable energy production method because hydrogen gas is renewable using the primary energy source, sunlight, and does not liberate carbon dioxide during combustion. The lower conversion efficiency of a biological system than to a solar battery could be compensated for by its lower requirement for energy and the lower initial investments. In this paper, we will review principles and recent progress in the development of photobiological hydrogen production methods, with reference to our works which were carried out in fiscal year 1991 to 1998 as a part of collaborative research with RITE (refer to Acknowledgement).
There are two kinds of production, hydrogenases types of photosynthesis, anoxygenic photosynthesis,
enzymes catalyzing hydrogen and nitrogenases, and two oxygenic photosynthesis and which can be carried out by
* Corresponding author. Present address: Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama-shi 701-1296, Japan.
bacteria, photobioreactor]
two groups of microorganisms, the cyanobacteria/green algae and the photosynthetic bacteria, respectively. We will review each combination of the enzyme and microorganism for hydrogen production. NITROGENASE-MEDIATED HYDROGEN PRODUCTION IN CYANOBACTERIA In 1974, Benemann and Weare (4) demonstrated that a nitrogen-fixing cyanobacterium, Anabaena cylindrica, produced hydrogen mediated by nitrogenase (5) and oxygen gas simultaneously in argon atmosphere for several hours. Nitrogenase mediates reduction of molecular nitrogen to ammonium with consumption of reducing power (mediated by ferredoxin) and ATP. However, nitrogenase can catalyze reduction of a proton in the absence of nitrogen gas (i.e., argon atmosphere). Furthermore, hydrogen production by nitrogenase enzyme occurs as a side reaction at the rate of one third to fourth of that of nitrogen fixation even in a 100% nitrogen gas atmosphere. The nitrogenase enzyme, itself, is extremely oxygenlabile. Cyanobacteria have a well-developed mechanism for the protection of nitrogenase from oxygen gas that can simultaneously supply both ATP and reducing power. The most successful strategy has been developed by a heterocystous and filamentous group of the organisms (6). Nitrogenase enzyme is localized in the heterocysts. Vegetative cells in filamentous cyanobacteria carry out oxygenic photosynthesis. Organic compounds produced by carbon dioxide reduction are transferred into heterocysts and decomposed to provide reducing power to nitrogenase. ATP can be provided by anoxygenic photosynthesis by Photosystem I in heterocysts. There have been several studies aimed at the prolongation and optimization of hydrogen production by nitrogen-fixing cyanobacteria (7-9).
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The physiological roles of ‘reversible’ hydrogenases in nitrogen-fixing cyanobacteria have been controversial, but ‘uptake’ hydrogenase is estimated to consume hydrogen gas for reutilization, which results in a decrease in the net hydrogen production (10). We reported aerobic hydrogen production (11) by a nitrogen-fixing Anabaena sp. (12) that was supposed to be an uptake hydrogenase-deficient strain. After 12d culture starting with a low inoculum concentration, the strain accumulated approximately 10% hydrogen and 70% oxygen gas in the gas phase of the vessel as products of the side reaction of nitrogenase. Recently, Hall and coworkers (13) also reported aerobic hydrogen production by using a Nostoc mutant that was deficient in ‘uptake’ hydrogenase. Miyamoto et al. (14) demonstrated outdoor hydrogen production by A. cylindrica in California. A nitrogenstarved culture of the cyanobacterium was continuously sparged by an argon-based gas mixture, and the hydrogen concentration in the effluent gas was measured. The average conversion efficiency over a one-month period (combustion energy of hydrogen gas produced by the cyanobacterium/incident solar energy in the photobioreactor area) was estimated to be approximately 0.2%. Mitsui and coworkers have extensively screened for cyanobacteria with high hydrogen productivity (15). Miami BG7, one of their most potent hydrogen producers was tested for outdoor culture (16). They isolated the unicellular and aerobic nitrogen-fixing Synechococcus sp. Miami BG0435 11, also and proposed a new strategy in unicellular and aerobic species to protect and drive oxygenlabile nitrogenase enzyme in oxygen-producing cells by developing synchronous culture techniques (17). This strain is a potent hydrogen producer. They estimated a conversion efficiency of 3.5% based on par (photosynthetically active irradiation that includes light energy of 400-700 mm in wavelength) using an artificial light source (18). HYDROGENASE-MEDIATED HYDROGEN PRODUCTION IN GREEN ALGAE AND CYANOBACTERIA Gaffron and Rubin (19) reported that a green alga, Scenedesmus, produces molecular hydrogen not only in the presence of light but also in the dark under anaerobic conditions. The enzyme mediating the hydrogen production was revealed to be hydrogenase (20, for review), catalyzing the reaction as follows: = HZ + 2Xoxidieed Z-I+ + &educed The electron carrier, X, is considered to be ferredoxin in green algae. Since ferredoxin is reduced with water during oxygenic photosynthesis, the green alga can be theoretically considered as being a water-splitting microorganism. In many cases, however, reducing power (electron donor) for hydrogenase is not always derived from the splitting of water but may partially from intracellular organic compounds such as starch in the presence of light. Green algae were considered as being light-dependent, water-splitting catalysts but hydrogen production using green algae was not feasible. Their hydrogenase enzyme is too oxygen-labile for sustainable hydrogen production: light-dependent hydrogen production ceases within minutes since the oxygen gas produced photosynthetically inhibits or inactivates hydroge-
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nase at less than 1% in the gas phase. A continuous gas flow system for maintaining a low oxygen concentration in a reaction vessel (21) seemed to be applicable for basic studies but for as a practical system. Seibert and coworkers (22) have been attempting to isolate mutants of Chlarnydomonas that posses an oxygen-tolerant hydrogenase. Green algae could be applied for another method of hydrogen production: use of dark and fermentative hydrogen production. Miura et al. (23-25) proposed hydrogen production in light/dark cycles. According to their proposal, carbon dioxide is reduced to starch by photosynthesis in the daytime and then the starch is decomposed to hydrogen gas, organic acids and alcohols under anaerobic conditions during the nighttime. We have reported that cyanobacteria can produce hydrogen in the dark under anaerobic conditions (2630). Spirulina species were demonstrated to have the highest hydrogen producing activities among those tested cm. The response by hydrogen-producing cyanobacterium, Mcrocystis aeruginosa to light is different to that of green algae (29). Light irradiation did not stimulate hydrogen production but resulted in hydrogen consumption. The light-dependent hydrogen uptake was not inhibited by the inhibitor of photosystem II, DCMU (3(3,Cdichloro-phenyl)-1,1-dimethyl urea), but inhibited by the antagonist of plastoquinone, DBMIB (2,5dibromo3-methyl-6-isopropyl-p-benzoquinone). These results suggest that hydrogen produced by degradation of endogenous glycogen may be taken up by the light-dependent reaction of photosystem I via plastoquinone. We partially purified a hydrogenase from A4. aeruginosa (31). The electron carrier for hydrogenase in cyanobacteria has still not been identified (30, 31). Recently, the genes cyanobacterial hydrogenases have cloned, and a new hypothesis relating to photosystem and the respiratory system was proposed (32). A NEW SYSTEM FOR HYDROGEN PRODUCTION: COMBINATION OF THE CYANOBACTERIAL PHOTOSYNTHETIC SYSTEM WITH CLOSTRIDIAL HYDROGENASE As described in the first section, some nitrogen-fixing cyanobacteria could be potential candidates for practical application to hydrogen production. Hydrogen production by nitrogenase, however, is an energy-consuming process due to hydrolysis of ATP molecules (at least 4 per 1 mol of hydrogen). On the other hand, hydrogenasemediated hydrogen production by cyanobacteria and green algae is economic due to the nonrequirement of ATP, although it is not sustainable under light conditions. The ideal and ultimate hydrogen producing system could be the sustainable hydrogenase-mediated, watersplitting system. Our strategy involves coupling of clostridial hydrogenase with the oxygenic photosynthetic system via ferredoxin (Fig. 1). To check the relevance of our strategy, in vivo coupling was tested; hydrogenase protein was electrointroduced into cyanobacterial cells (33). The cyanobacterial cells that contained clostridial hydrogenase and then treated with protease produced hydrogen gas upon light irradiation. Then, we attempted to overexpress the hydrogenase in a cyanobacterium, Synechococcus PCC7942, by developing a genetic engineering system (34-37). The adjustment
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FIG. 1. Schematic of photosynthetic (A) and transformant (B) cyanobacteria.
electron flows in wild-type
of the Shine-Dalgarno sequence to the host cyanobacteriurn was supposed to be critical for heterologous expression (38). Finally, we succeeded in heterologous expression of the hydrogenase (Asada et al., in submission) which was thought to be very difficult since the hydrogenase gene is composed of many accessory gene elements (10). We are presently characterizing the transformant strain, particularly in relation to its genetically introduced hydrogenase activity. HYDROGEN PRODUCTION BY PHOTOSYNTHETIC BACTERIA
Photosynthetic bacteria carry out anoxygenic photosynthesis using organic compounds and reduced sulfur compounds as electron donors, which are categorized as non-sulfur and sulfur photosynthetic bacteria, respectively. Some non-sulfur photosynthetic bacteria are potent hydrogen producers utilizing organic acids such as lactic, succinic and butyric acids or alcohols. Since light energy is not used for water oxidation, the efficiency of light energy for the production of hydrogen by photosynthetic bacteria is theoretically much higher than that by cyanobacteria. From the practical point of view, photosynthetic bacteria are important since they can be used for dual purposes of waste water treatment and hydrogen production (39, 40). Recycling of agricultural wastes and various biomasses has been also realized simultaneously with hydrogen production by photosynthetic bacteria. Ike et al. demonstrated that raw algal biomass can be used as a resource for hydrogen production. They proposed chemical digestion of the algal mass, to produce good substrates for photosynthetic bacteria from starch, a major constituent of algal biomass, to improve the yield of hydrogen (41). They also demonstrated fermentation of living algal biomass by a lactic acid bacterium (42-44). Lactic acid is an excellent substrate for hydrogen production by photosynthetic bacteria, and a maximum hydrogen yield of 8 mol/mol starch-glucose from algal biomass was observed. The enzyme catalyzing hydrogen production by photosynthetic bacteria is nitrogenase (5), whereas hydrogenases may be active for hydrogen uptake in many photosynthetic bacteria. Miyake and Kawamura (45) demonstrated that 6 to 8% of the incident light energy can be converted to hydrogen gas (combustion energy) in laboratory experiments using our isolate, Rhodobacter sp. 8703 (46) with lactic acid as an electron donor.
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Anaerobic bacteria degrade sugars to hydrogen gas and organic acids (47, i.e.), but cannot utilize the organic acids as electron donors. Miyake et al. (48) demonstrated the combined use of photosynthetic bacteria with anaerobic bacteria for complete conversion of sugar to hydrogen. Theoretically, 1 mol of glucose can be converted to 12mol of hydrogen, and their experimental result was 7 mol of hydrogen per mole of consumed glucose. Carbohydrate polymers such as starch or cellulose, the major constituents of agricultural wastes and biomasses, are rarely degraded by photosynthetic bacteria alone for hydrogen production. Ike et al. proposed a one-step hydrogen production from starch using the halophilic bacterial community originating from activated sludge (49, 50). In this community, starch could be converted to hydrogen directly by the cooperative action of Rhodobium marinum and Vibrio Jluvialis. From the practical point of view, organic wastes frequently contain sugar or sugar polymers. The combined use of the two kinds of bacteria would enhance their probability of application in photobiological hydrogen production (51). GENETIC ENGINEERING TO CONTROL EXPRESSION OF THE PHOTOSYNTHETIC PROTEINS
For efficient hydrogen production, the balance between a photosystem and hydrogen producing enzyme is required. For cyanobacteria, overexpression of heterologous hydrogenase was attempted as described above. Compared to nitrogenase activity in photosynthetic bacteria, too much light energy is converted to biochemical energy by the photosystem, which is lost via other biochemical processes. We attempted to reduce the photosystem content to an appropriate level for the present nitrogenase activity. We have developed a new genetic engineering method to control the expression of photosynthetic protein complexes; the promoter of the puf operon from Rhodobacter sphaeroides RV encoding the photoreaction center and light-harvesting proteins (52) was introduced into the host to absorb active ribosomes to prevent transcription of the puf operon. Furthermore, we have obtained by UV irradiation a mutant, P3, with altered composition of light-harvesting complexes (53). Hydrogen production by the P3 mutant was characterized by using a hydrogen electrode system under monochromatic light of several wavelengths. When 800 nm and 850nm monochromatic light was irradiated, the mutant strain produced hydrogen gas at a higher rate than the wild type (Fig. 2). These results suggest that the composition of lght-harvesting proteins may finally affect the productivity of hydrogen gas. RESEARCH AND PHOTOBIOREACTORS HYDROGEN
DEVELOPMENT OF FOR PHOTOBIOLOGICAL PRODUCTION
‘Self-shadowing’ has posed a difficult problem with regard to the utilization of solar energy by cultures of photosynthetic microorganisms. Quite a small portion of photobioreactors is actually active due to the inability of the light penetrate deeply when microbial cell concen-
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Light intensity, W/m2, SOOnm
’ 0
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Light intensity, W/m2,850nm
0
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Light intensity, W/m2,875nm
FIG. 2. Comparison of hydrogen production rates by the wild-type and P3 mutant strain of Rb. sphueroides RV (55). Symbols: 0, wild strain; 0, P3 mutant. (a) 800 nm; (b) 850 nm; (c) 875 nm. Refer (55) for detailed experimental conditions.
tration is high. Therefore, a basic analysis on the distribution of light intensity and shift in composition wavelength in relation to hydrogen production (55-57) is important. Wakayama et al. studied on effects of chopped light on hydrogen production by photosynthetic bacteria (58). Moreover, immobilization of bacteria could contribute to prolongation, stabilization and ease in exchange of culture broth. We have developed several immobilization methods using glass plates, glass beads and agar gels (59-62). A photobioreactor for hydrogen production by cyanobacteria was studied by Markov et al. (63). Various types of photobioreactors in RITE Hydrogen Project (refer to Acknowledgments) has been demonstrated (6467). The largest photobioreactor (Fig. 3) developed for hydrogen production is a floating type (4001 working volume) (67).
The future and technological applicability of biological hydrogen production depends not only on progress in research, i.e., improvement of efficiency by breeding or developing bioreactor, but also on economical factors such as of fossil fuels, and social acceptance and development of hydrogen energy systems. ACKNOWLEDGMENTS Our studies were partially performed as a part of R & D Projects of Environmentally-Friendly Technology for the Production of Hydrogen under the management of RITE (Research Institute of Innovative Technologies for the Earth) which was sponsored by MITI (Ministry of International Trade and Industry) with the supports of the New Energy and Industrial Technology Development Organization (NEDO). REFERENCES
FUTURE PROSPECTS We consider that biological hydrogen production is the most challenging undertaking issue in the field of biotechnology for overcoming environmental problems.
1. Miyamoto, K.: Hydrogen production by photosynthetic bacteria and microalgae, p. 771-786. In Murooka, Y. and Imanaka, T. (ed.), Recombinant microbes for industrial and agricultural applications. Marcel Dekker Inc., New York, Base], and Hong Kong (1993). 2. Sasikala, K., Ramaoa, C. H., Rao, P. R., and Kovacs, K. L.: Anoxygenic phototrophic bacteria: physiology and advances in hydrogen production technology. Adv. Appl. Microbial., 38, 211-295 (1993). 3. Miyake, J.: Photosynthetic bacteria for solar energy conversion, p. 1019-1025. In Alberghina, L., Frontali, L., and Sensi, P. (ed.), ECBB: Proceeding of the 6th European Congress on Biotechnology. Elsevier, Amsterdam (1994). 4. Benemann, J.R. and Weare, N. M.: Hydrogen evolution by nitrogen-fixing Anabuena cylindrica culture. Science, 184, 174175 (1974). J., Asada, Y., and Kewamura, S.: Nitrogenase, p. 5. Miyake, 362-370. In Kitani, 0. and Hall, C. W. (ed.), Biomass handbook. Gordon and Breach Science Publishers, New York (1989). 6. Wolk, C. P., Ernst, A., and Elhai, J.: Heterocyst metabolism and development, p. 769-823. In Bryant, D.A. (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht (1994). 7 Weissman, J. C. and Renemann, J. R.: Hydrogen production by nitrogen-fixing cultures of Anabaean cylindrica. Appl. Environ. Microbial., 33, 123-131 (1977). 8. Miyamoto, K., Benemann, J. R., and Haltenbeck, P. C.: Nitrogen fixation by thermophilic blue-green algae (cyanobacteria): temperature characteristics and potential use in biophotolysis. I
FIG. 3. A floating type of photobioreactor (67). Hydrogen production was carried out under natural sunlight.
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Appl. Environ. Microbial., 37, 454-458 (1979). 9. Asada, Y., Tomizulca, N., and Kawamura, S.: Prolonged hydrogen evolution by a cyanobacterium (blue-green alga), Anabaena sp. J. Ferment. Technol., 63, 85-90 (1985). 10. Hancel, A. and Lindblad, P.: Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen a clean and renewable energy source. Appl. Microbial. Biotechnol., 50, 153-160 (1998). 11. Asada, Y. and Kawamura, S.: Aerobic hydrogen accumulation by a nitrogen-fixing cyanobacterium, Anabaena sp. Appl. Environ. Microbial., 51, 1063-1066 (1986). 12. Asada, Y., Tonomura, K., and Nakayama, 0.: Hydrogen evolution by an isolated strain of Anabuenu. J. Ferment. Technol., 57, 28&286 (1979). 13. Lichtel, R., Basin, J., and Hall, D. 0.: The biotechnology of hydrogen production by Nostoc flugelliforme grown under chemostat conditions. Appl. Microbial. Biotechnol., 47, 701707 (1997). 14. Miyamoto, K., Benemann, J. R., and Hallenbeck, P. C.: Solar energy conversion by nitrogen limited cultures of Anubuenu cylindricu. J. Ferment. Technol., 57, 287-293 (1979). 15. Kumazawa, S. and Mitsui, A.: Characterization and optimization of hydrogen photoproduction by a salt water blue-green alga, Oscilkztoriu sp. Miami BG7. I. Enhancement through limiting the supply of nitrogen nutrients. Int. J. Hydrogen Energy, 6, 339-348 (1981). 16. Phlips, E. J. and Mitsui, A.: Development of hydrogen production activity in the marine blue-green alga, Oscillutoriu sp. Miami BG7 under natural sunlight conditions, p. 801-804. In Sysbesma, C. (ed.), Advance in Photosynthetic Research, vol. 2. Martiuus Nijhoff/Dr. W. Junk Publishers, Hague (1984). 17. Mitsui, A., Kumazawa, S., Takahashi, A., Ikemoto, H., Cao, S., and Arai, T.: Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature, 323, 720722 (1986). 18. Kumazawa, S. and Mitsui, A.: Efficient hydrogen photoproduction by synchronously grown cells of a marine cyanobacterium, Synechococcus sp. Miami BG 043511, under high cell density conditions. Biotech. Bioeng., 44, 854-858 (1994). 19. Gaffron, H. and Rubin, J.: Fermentative and photochemical production of hydrogen in algae. J. Gen. Physiol., 26, 219-240 (1942). 20. Schulz, R.: Hydrogenases and hydrogen production in eukaryotic organisms and cyanobacteria. J. Marine Biotech., 4, 16-22 (1996). 21. Greenbaum, E.: Energetic efficiency of hydrogen photoevolution by algal water splitting. Biophysical. J., 54, 365-368. (1988). 22. Seibert, M., Flynn, T., Benson, D., Tracy, E., and Ghirardi, M.: Development of selected and screening procedures for rapid identification of Hz-producing algal mutants with increased O2 tolerance, p. 227-234. In Zaborsky, 0. R. (ed.), Biohydrogen, Plenum Press, London (1998). 23. Miura, Y., Ohta, S., Mano, M., Miyamoto, K.: Isolation and characterization of unicellular marine green alga exhibiting high activity in the dark hydrogen production. Agr. Biol. Chem., 50, 2837-2844 (1986). 24. Miura, Y., Matsuoka, S., Miyamoto, K., and Saitoh, C.: Stably sustained hydrogen production with high molar yield through a combination of a marine green alga and a photosynthetic bacterium. Biosci. Biotech. Biochem., 56, 751-754 (1992). 25. Miura, Y., Akano, T., Fukatsu, K., and Miyasaka, H.: Hydrogen production by a combination of a marine green alga and a photosynthetic bacterium, p. 42. In Proc. RITE Int. Workshop on Biological Hydrogen production (24-25 Nov., Tokyo, Japan) (1994). 26. Asada, Y. and Kawamura, S.: Hydrogen evolution by Microcystis ueruginosu in darkness. Agric. Biol. Chem., 48, 2595-2596 (1984). 27. Asada, Y. and Kawamura, S.: Hydrogen evolving activity among the genus, Microcystis, under dark and anaerobic conditions. Rept. Ferment. Res. Inst., 63, 39-54 (1984).
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