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Effect of partial shading on photoproduction of hydrogen by Chlorella
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 97, No. 5, 317–321. 2004
Effect of Partial Shading on Photoproduction of Hydrogen by Chlorella EIICHI KOJIMA1* AND BIN LIN1 Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan1 Received 26 August 2003/Accepted 25 February 2004
The photoproduction of hydrogen by the green alga Chlorella pyrenoidosa was studied in 650ml bubble columns made of glass with 7 cm diameter. Hydrogen was produced by adding sodium dithionite as an oxygen scavenger directly to an algal suspension. When a part of the wall of the bubble column was shaded with a sheet of plastic film impervious to light, the production rate and total volume of hydrogen increased as compared to those in the columns without partial shading. This relationship between the hydrogen photoevolution rate and the proportion of lighted region is contrary to normal photochemical reactions. This phenomenon is considered to be related to the regulation by light of the activity of the enzymes and/or the photosynthetic electron transport systems, which was examined by measuring fluorescence induction curves of dark-adapted Chlorella cells and also the distribution of light intensity within the bubble column bioreactors. [Key words: microalga, Chlorella pyrenoidosa, hydrogen production, photobioreactor, bubble column]
effects of partial shading of the wall of the glass bubble columns, by which the activity of the light-regulated enzymes could be controlled, on the production rate and amount of hydrogen. During the hydrogen photoevolution, fluorescence induction curves were obtained for dark-adapted Chlorella cells, and the relationship between the hydrogen evolution rate and the lighted volume ratio of algal suspension is discussed.
Hydrogen production by microalgae has been studied as a potential means of providing renewable energy. Microalgae can grow autotrophically using CO2, and can be used for fixing CO2 at major sources of the gas such as in power plants that consume fossil fuels. Two mechanisms of photoproduction of hydrogen by green algae have been proposed. One involves biophotolysis of water, which requires abundant water as an electron donor, but the concomitantly produced oxygen must be removed to circumvent the inactivation of hydrogenase. For this purpose oxygen absorbers (1–4) or inert carrier gases have been applied (5–8). The concentration of oxygen could be reduced sufficiently during photoevolution of hydrogen by a green alga, after sulfur deprivation was imposed upon the cells, and coordinated photosynthetic and respiratory electron transport were achieved (9, 10). The other involves reduction of the plastoquinone pool with endogenous reductant (11), enabling temporal or spatial separation of hydrogen evolution and oxygen evolution (12, 13). Under moderate temperature and irradiance, the main sink of photosynthetically generated electrons that pass through ferredoxin is the Calvin cycle. However, under low incident light intensity (1–3 W m–2) or low temperature (ca. 0°C), the electrons are partitioned between the two competitive pathways of hydrogen evolution and the Calvin cycle, the former being predominant (6), and the partitioning ratio being controllable to a certain extent (7). Generally, when the concentration of algal cells is high or the culture vessel is large, the light intensity within the vessel is reduced and the rate of hydrogen photoevolution is affected. The objective of this study was to examine the
MATERIALS AND METHODS Culture conditions Chlorella pyrenoidosa C-101 was obtained from the IAM Culture Collection of the Institute of Molecular and Cellular Biosciences, University of Tokyo. Modified Bristol medium supplemented with 1 g/l proteose peptone was used as the culture medium. Precultures were first grown in a 50-ml oblong flat flask, then in a 3-l conical flask to desired cell concentrations. Cultures were gassed with 5% CO2 in air at 25°C and continuously illuminated with a bank of six 10 W fluorescent lamps set around the flask. The algal suspension was transferred to bubble column bioreactors made of glass, 650 ml in liquid volume, 7 cm in diameter with conical part 8.5 cm in height at the bottom. For induction of hydrogenase, the columns were gassed with N2 in the dark for more than 20 h. Hydrogen photoevolution Before the start of illumination, sodium dithionite (10 mM) was added as an oxygen scavenger directly to the algal suspension. Illumination was provided by a bank of four 20 W fluorescent lamps placed on one side of the bubble columns, and the light intensity at the inner wall of the illuminated side was 6500 lx (94 mmol s–1m–2). During hydrogen photoevolution, the algal culture was gassed with nitrogen at 150 ml/min, and the effluent gas was analyzed by gas chromatography (using Shimadzu GC-3BT; Shimadzu, Kyoto). In the case of the experiment involving partial shading, sheets of plastic film impervious to light were fixed on the wall. The degree of shading was expressed as the ratio of lighted volume VL to the total volume of algal sus-
* Corresponding author. e-mail: [email protected] 317
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pension. Fluorescence measurement Fluorescence was measured at 685 nm with a laboratory-constructed fluorometer as described in previous papers (3, 14), and the intensity of actinic light which passed through blue and heat-absorbing filters was set at 25 mmol s–1m–2. The algal concentration was diluted with a buffer solution to 0.2 g/l, and the algal cells were dark-adapted for a specified period up to 10 min under the atmosphere of nitrogen gas. The output of the fluorometer was recorded with a personal computer equipped with an analogue-to-digital converter. Hydrogenase activity Methyl viologen (6 mM), sodium dithionite and 0.1 M phosphate buffer were added to the pellet of algal cells after centrifugation under nitrogen atmosphere, and the suspension was incubated for a specified reaction time (20 to 30 min) in the dark. The hydrogenase activity was measured as the rate of hydrogen evolution from reduced methyl viologen. Light intensity distribution The distribution of light intensity within the bubble column was measured with a diffusing sphere photoprobe as described previously (3, 15) , which was made of a polymethylmethacrylate resin sphere 3.2 mm in diameter and plastic fiber optics.
RESULTS Effect of shading on the rate of hydrogen photoevolution The rates of hydrogen photoevolution are shown in Fig. 1. At high cell concentrations, the second peak of hydrogen evolution was observed 20 h after the start of illumination, the width of the second peak being much broader than the initial peak (Fig. 1A). When the column wall was not covered with a sheet of film impervious to light (Fig. 1A, B, open circles), a distinct initial gush of hydrogen was observed shortly after the start of illumination, while the initial peak of hydrogen was low or completely disappeared when a part of the column was covered (Fig. 1B, C, solid circles). The most noticeable effect of the film was that, except the initial peaks, the rate of photoevolution of hydrogen was higher in the cases with partial shading than without shading, the effect being more noticeable under the conditions of lower cell concentrations. Figure 2 shows the hydrogen evolution rate after the initial peak, i.e., that at the steady state or the top of the second peak. The lowest VL shown in Fig. 2, 0.17, means that the whole area of the wall of the glass column except the conical bottom was covered with the film. The rates of hydrogen photoevolution were higher at lower ratios of lighted volume VL. This dependence of hydrogen photoevolution rate on the lighted volume is contrary to that in normal photochemical reactions, where the overall reaction rates increase with the lighted volume ratio. Chlorophyll a fluorescence induction curves Figure 3 shows the variation with time in the chlorophyll a fluorescence induction curves measured after 5 min dark adaptation, which correspond to the course of H2 production shown in of Fig. 1A. While the rates of hydrogen photoevolution were sustained at a high level, the fluorescence induction curves showed characteristic variations corresponding to the phases named I, D, P, and S (14, 16, 17). The I-peak is the highest point in the induction curve, which is the same phenomenon reported for Scenedesmus cells kept under dark and anaerobic conditions, and the I-peak is caused by inactivation of photosystems in the dark and their reactiva-
FIG. 1. Hydrogen photoevolution rate and hydrogenase activity with and without partial shading. Algal cell concentrations: (A) 1.3 and 1.4 kg/m3; (B) 0.69 and 0.75 kg/m3; (C) 0.45 and 0.46 kg/m3. Values of VL in hydrogenase activity data: open squares, 1.0; solid squares, 0.17.
FIG. 2. Hydrogen photoevolution rate as a function of algal cell concentration.
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FIG. 5. Total hydrogen volume produced as a function of algal cell concentration. FIG. 3. Fluorescence induction curves in the course of hydrogen photoevolution shown in Fig. 1A. The figures at the top of the graph indicate the time at which each algal sample was obtained. Values of VL: open circles, 1.0; solid circles, 0.17.
FIG. 6. Cross-sectional distribution of light intensity within the bubble column photobioreactors filled with algal cell suspension. FIG. 4. Effect of lighted volume ratio (VL) and dark adaptation periods (t) on fluorescence induction curves during the steady hydrogen photoevolution. Algal cell concentration: 0.7 kg/m3. (A) VL = 1.0 [-]; (B) VL = 0.47 [-]; (C) VL = 0.17 [-].
tion by actinic light (17–19). The P-peak, which was observed about 5 s after the illumination of the actinic light, reflects the reactivation of inactivated ferredoxin-NADP+ reductase by the actinic light and the consequent resumption of electron flow through the Calvin cycle (20, 21). In Fig. 3, the heights of the I- and P-peaks decreased as the rate of hydrogen evolution gradually decreased, indicating that the mediating electron carriers were gradually damaged, and that the partial shading serves to preserve the initial shape of fluorescence induction curves, i.e., the initial photosynthetic activity is maintained over a longer period by partial shading. Figure 4 shows the fluorescence induction curves during the steady phase of H2 photoevolution under experimental conditions similar to that in Fig. 1B. The induction curves were obtained for three different VL values and for two different dark adaptation periods after sampling from the photobioreactor (t = 30 s and 10 min). The induction curves for
t = 10 min are similar to those in Fig. 3, while the heights of the I- and P-peaks for t = 30 s are lower than those for t = 10 min. These characteristics will be discussed later. Total hydrogen volume Figure 5 shows the total hydrogen volume photoproduced as functions of algal cell concentration. Although the total volume of hydrogen increased with decreasing ratio of the lighted area, the difference in the total volume with and without the films decreased as the cell concentration increased. The higher total hydrogen volume in cases with partial shading is due to the combined effects of the increase in the hydrogen evolution rate and the preservation of the photosystems including the activity of hydrogenase. The maximum total hydrogen volume for VL = 0.17 was 6.9 ´10–2 m3/kg at the cell concentration of 0.6 kg/m3, which is 1.5 times higher than for the cases without partial shading. Light intensity distribution within the culture vessel Figure 6 shows cross-sectional distributions of light intensity within the bubble column filled with algal suspension. The values shown in Fig. 6 represent the relative light intensity expressed as percentages of the reference intensity (6500 lx), which was measured at the inside wall of the
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FIG. 7. Light intensity distribution on the axis of bubble column photobioreactors with and without partial shading. Algal cell concentrations: (A) 0.26 kg/m3; (B) 0.73 kg/m3; (C) 0.96 kg/m3; (D) 1.29 kg/m3.
vessel filled with distilled water. The relative light intensity distributions on the axis of the column are shown in Fig. 7, where the regions of the depth larger than 14 cm with high relative light intensity correspond to the conical part of the bubble column bioreactor. On the basis of the cross-sectional and axial light intensity distribution, the volume ratio of the lighter region (to the total culture volume), where the relative light intensity is higher than 50% (3250 lx) or 28% (1820 lx) of the reference intensity, was calculated (Fig. 8). For instance, at a cell concentration of 1 g/l, the volume lighter than 1820 lx is 45% of the total suspension volume at VL = 1.0 (Fig. 8A), while it is only 7% at VL = 0.17 (Fig. 8C). The results in Fig. 8 as well as in Fig. 2 lead to the conclusion that the highest rate of hydrogen photoevolution was obtained when a large part (more than 90%) of the algal suspension was kept under low illumination of darker than 1800 lx. DISCUSSION The rate of hydrogen photoevolution increased as the volume ratio of the lighted region decreased (Fig. 2). This relationship between the area of the lighted region and the reaction rate is contrary to normal photochemical reactions. A possible explanation for this phenomenon is that the partitioning of electrons between the hydrogen photoevolution pathway via hydrogenase and the Calvin cycle (6, 7) is involved in the following manner. As described in Fig. 3, the I- and P-peaks reflect the rapid reactivation of photosystems and the regulation of electron flow via the ferredoxin-
FIG. 8. Volume ratio of lighter region (to the total culture volume) where the relative light intensity is higher than 50% or 28% of reference intensity (6500 lx). (A) VL = 1.0 [-]; (B) VL = 0.47 [-]; (C) VL = 0.17 [-].
NADP+ reductase (FNR), respectively, both by actinic light. In Fig. 4, the fluorescence induction curves obtained for t = 10 min show characteristic peaks of I and P of dark-adapted algal cells (Figs. 4A-2, B-2, C-2). On the other hand, the shapes of the induction curves for t = 30 s are determined by the illumination conditions for the bioreactors, i.e., the curves correspond to photosynthetic activities of the cells adapted to different light conditions within the reactors. When the lighted volume ratio is low (Fig. 4C-1), distinct I- and Ppeaks are observed as in the case of t = 10 min, indicating that dark-adapted cells are predominant in the photobioreactor. On the other hand, both peaks are less distinct at higher lighted volume ratios, corresponding to higher proportions of light-adapted cells with reactivated photosystems (Fig. 4A-1, B-1). On the basis of these observations, the inverse relation between the lighted volume ratio and hydrogen photoevolution rate can be explained as follows. If a part of the column wall is covered with the film impervious to light, the photosystems and FNR are inactivated while they are carried into the dark region by the liquid flow. When these dark-adapted cells are carried into the lighted region again, electron flow via the ferredoxin is partitioned mainly to light-driven hydrogen evolution during the lag time (about 5 s) before FNR is reactivated (6, 21). Although the electrons are supplied also to the Calvin cycle after the
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reactivation of the FNR, and consequently the rate of hydrogen photoevolution could be reduced, if the ratio of the lighted region is properly low, the algal cells with high hydrogen production rate will be predominant in the lighted region, and the overall light-driven hydrogen production rate could be kept at a higher value for the columns with partial shading. The effects of active oxygens on H2 photoevolution are discussed from the viewpoint of damage to photosystems or hydrogenase. As described in Fig. 3, damage to photosystems is not apparent while the H2 photoevolution rates were sustained at a high level, which indicates that the lower hydrogen evolution rates observed in the higher VL conditions are not due to the damage in photosystems. As for damage to hydrogenase, the hydrogenase activity remained relatively stable even after cessation of H2 photoevolution (Fig. 1C). If it is assumed that the rate of H2 photoevolution is proportional to the product of the hydrogenase activity and VL value, at 23 h after the start of illumination, as shown in Fig. 1C, the ratio of H2 evolution rate with partial shading to that without shading would be approximately (4.7 ´10–7 ´0.17)/ (2.7 ´10–7 ´1.0) = 0.3. The actual ratio at the same time point is (1.3´10–10)/(0.48´10–10)=2.7. This indicates that although the hydrogenase activity might be partly inhibited by active oxygens, the high values of H2 evolution rate for the partial shading conditions could be explained by the kinetic behavior of FNR, i.e., decrease in the proportion of electrons partitioned to the Calvin cycle prior to the reactivation of FNR in the light. Finally, the effect of light activation of the Calvin cycle enzymes on H2 photoevolution is briefly discussed. While electrons from photosystem I are transferred via ferredoxin to the light-regulated FNR as reducing power for the synthesis of carbohydrates, a part of the electrons from ferredoxin is transmitted to ferredoxin–thioredoxin reductase (FTR) as a regulatory electron signal to activate the Calvin cycle enzymes in the light (22, 23). In the above discussion, the regulation of hydrogen photoevolution rate is assumed to result from the linear electron transport via FNR and not from the electron signal via FTR. In conclusion, this study demonstrates that the photobioreactors with proper partial shading are better suited for hydrogen production in that the evolution rate is higher and the photosystems or necessary enzymes are better preserved. REFERENCES 1. Pow, T. and Krasna, A. I.: Photoproduction of hydrogen from water in hydrogenase-containing algae. Arch. Biochem. Biophys., 194, 413–421 (1979). 2. Rosenkrans, M. and Krasna, A. I.: Stimulation of hydrogen photoproduction in algae by removal of oxygen by reagents that combine reversibly with oxygen. Biotechnol. Bioeng., 26, 1334–1342 (1984). 3. Kojima, E. and Yamaguchi, Y.: Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa. J. Ferment. Technol., 66, 19–25 (1988). 4. Kojima, E.: Effect of inorganic salts on the duration of hydrogen photoevolution by Chlorella. Kagaku Kogaku Ronbun-
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shu, 19, 908–913 (1993). 5. Greenbaum, E.: Simultaneous photoproduction of hydrogen and oxygen by photosynthesis. Biotechnol. Bioeng. Symp., No. 10, 1–13 (1980). 6. Graves, D. A., Tevault, C. V., and Greenbaum, E.: Control of photosynthetic reductant: the role of light and temperature on sustained hydrogen photoevolution by Chlamydomonas sp. in an anoxic, carbon dioxide-containing atmosphere. Photochem. Photobiol., 50, 571–576 (1989). 7. Cinco, R. M., MacInnis, J. M., and Greenbaum, E.: The role of carbon dioxide in light-activated hydrogen evolution by Chlamydomonas reinhardtii. Photosynth. Res., 38, 27–33 (1993). 8. Greenbaum, E., Blankinship, S. L., Lee, J. W., and Ford, R. M.: Solar photobiochemistry: simultaneous photoproduction of hydrogen and oxygen in a confined bioreactor. J. Phys. Chem. B, 105, 3605–3609 (2001). 9. Melis, A., Zhang, L., Forestier, M., Ghirardi, M. L., and Seibert, M.: Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol., 122, 127–135 (2000). 10. Melis, A. and Happe, T.: Hydrogen production. Green algae as a source of energy. Plant Physiol., 127, 740–748 (2001). 11. Bamberger, E. S., King, D., Erbes, D. L., and Gibbs, M.: H2 and CO2 evolution by anaerobically adapted Chlamydomonas reinhardtii F-60. Plant Physiol., 69, 1268–1273 (1982). 12. Ike, A., Kawaguchi, H., Hirata, K., and Miyamoto, K.: Hydrogen photoproduction from starch in algal biomass, p. 53– 61. In Miyake, J., Matsunaga, T., and San Pietro, A. (ed.), Biohydrogen II. Elsevier Science, Oxford (2001). 13. Miura, Y., Yagi, K., Shoga, M., and Miyamoto, K.: Hydrogen production by a green alga Chlamydomonas reinhardtii in an alternating light/dark cycle. Biotechnol. Bioeng., 24, 1535–1563 (1982). 14. Katoh, S.: Keikou sokutei (fluorescence measurements), p. 205–225. Gakkai Shuppan Center, Tokyo (1983). (in Japanese) 15. Tanaka, M., Itaya, A., and Okamoto, K.: Determination of light intensity profiles in gas and liquid dispersions by use of diffusing sphere photoprobe. Kagaku Kogaku Ronbunshu, 10, 551–556 (1984). 16. Hipkins, M. F. and Baker, N. R.: Photosynthesis energy transduction, p. 90–92. IRL Press, Oxford (1986). 17. Schreiber, U. and Vidaver, W.: Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus. Biochim. Biophys. Acta, 368, 97–112 (1974). 18. Kessler, E.: Effect of hydrogen adaptation on fluorescence in normal and manganese-deficient algae. Planta, 81, 264–273 (1968). 19. Kessler, E.: Effect of anaerobiosis on photosynthetic reactions and nitrogen metabolism of algae with and without hydrogenase. Arch. Mikrobiol., 93, 91–100 (1973). 20. Satoh, K. and Katoh, S.: Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: the site of light-dependent regulation of electron transport. Plant Cell Physiol., 21, 907–916 (1980). 21. Satoh, K.: Fluorescence induction and activity of ferredoxinNADP+ reductase in Bryopsis chloroplasts. Biochim. Biophys. Acta, 638, 327–333 (1981). 22. Schürmann, P. and Jacquot, J.-P.: Plant thioredoxin systems revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol., 51, 371– 400 (2000). 23. Balmer, Y., Koller, A., del Val, G., Manieri, W., Schürmann, P., and Buchanan, B. B.: Proteomics gives insight into the regulatory function of chloroplast thioredoxins. Proc. Natl. Acad. Sci. USA, 100, 370–375 (2003).
Effect of Partial Shading on Photoproduction of Hydrogen by Chlorella EIICHI KOJIMA1* AND BIN LIN1 Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan1 Received 26 August 2003/Accepted 25 February 2004
The photoproduction of hydrogen by the green alga Chlorella pyrenoidosa was studied in 650ml bubble columns made of glass with 7 cm diameter. Hydrogen was produced by adding sodium dithionite as an oxygen scavenger directly to an algal suspension. When a part of the wall of the bubble column was shaded with a sheet of plastic film impervious to light, the production rate and total volume of hydrogen increased as compared to those in the columns without partial shading. This relationship between the hydrogen photoevolution rate and the proportion of lighted region is contrary to normal photochemical reactions. This phenomenon is considered to be related to the regulation by light of the activity of the enzymes and/or the photosynthetic electron transport systems, which was examined by measuring fluorescence induction curves of dark-adapted Chlorella cells and also the distribution of light intensity within the bubble column bioreactors. [Key words: microalga, Chlorella pyrenoidosa, hydrogen production, photobioreactor, bubble column]
effects of partial shading of the wall of the glass bubble columns, by which the activity of the light-regulated enzymes could be controlled, on the production rate and amount of hydrogen. During the hydrogen photoevolution, fluorescence induction curves were obtained for dark-adapted Chlorella cells, and the relationship between the hydrogen evolution rate and the lighted volume ratio of algal suspension is discussed.
Hydrogen production by microalgae has been studied as a potential means of providing renewable energy. Microalgae can grow autotrophically using CO2, and can be used for fixing CO2 at major sources of the gas such as in power plants that consume fossil fuels. Two mechanisms of photoproduction of hydrogen by green algae have been proposed. One involves biophotolysis of water, which requires abundant water as an electron donor, but the concomitantly produced oxygen must be removed to circumvent the inactivation of hydrogenase. For this purpose oxygen absorbers (1–4) or inert carrier gases have been applied (5–8). The concentration of oxygen could be reduced sufficiently during photoevolution of hydrogen by a green alga, after sulfur deprivation was imposed upon the cells, and coordinated photosynthetic and respiratory electron transport were achieved (9, 10). The other involves reduction of the plastoquinone pool with endogenous reductant (11), enabling temporal or spatial separation of hydrogen evolution and oxygen evolution (12, 13). Under moderate temperature and irradiance, the main sink of photosynthetically generated electrons that pass through ferredoxin is the Calvin cycle. However, under low incident light intensity (1–3 W m–2) or low temperature (ca. 0°C), the electrons are partitioned between the two competitive pathways of hydrogen evolution and the Calvin cycle, the former being predominant (6), and the partitioning ratio being controllable to a certain extent (7). Generally, when the concentration of algal cells is high or the culture vessel is large, the light intensity within the vessel is reduced and the rate of hydrogen photoevolution is affected. The objective of this study was to examine the
MATERIALS AND METHODS Culture conditions Chlorella pyrenoidosa C-101 was obtained from the IAM Culture Collection of the Institute of Molecular and Cellular Biosciences, University of Tokyo. Modified Bristol medium supplemented with 1 g/l proteose peptone was used as the culture medium. Precultures were first grown in a 50-ml oblong flat flask, then in a 3-l conical flask to desired cell concentrations. Cultures were gassed with 5% CO2 in air at 25°C and continuously illuminated with a bank of six 10 W fluorescent lamps set around the flask. The algal suspension was transferred to bubble column bioreactors made of glass, 650 ml in liquid volume, 7 cm in diameter with conical part 8.5 cm in height at the bottom. For induction of hydrogenase, the columns were gassed with N2 in the dark for more than 20 h. Hydrogen photoevolution Before the start of illumination, sodium dithionite (10 mM) was added as an oxygen scavenger directly to the algal suspension. Illumination was provided by a bank of four 20 W fluorescent lamps placed on one side of the bubble columns, and the light intensity at the inner wall of the illuminated side was 6500 lx (94 mmol s–1m–2). During hydrogen photoevolution, the algal culture was gassed with nitrogen at 150 ml/min, and the effluent gas was analyzed by gas chromatography (using Shimadzu GC-3BT; Shimadzu, Kyoto). In the case of the experiment involving partial shading, sheets of plastic film impervious to light were fixed on the wall. The degree of shading was expressed as the ratio of lighted volume VL to the total volume of algal sus-
* Corresponding author. e-mail: [email protected] 317
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pension. Fluorescence measurement Fluorescence was measured at 685 nm with a laboratory-constructed fluorometer as described in previous papers (3, 14), and the intensity of actinic light which passed through blue and heat-absorbing filters was set at 25 mmol s–1m–2. The algal concentration was diluted with a buffer solution to 0.2 g/l, and the algal cells were dark-adapted for a specified period up to 10 min under the atmosphere of nitrogen gas. The output of the fluorometer was recorded with a personal computer equipped with an analogue-to-digital converter. Hydrogenase activity Methyl viologen (6 mM), sodium dithionite and 0.1 M phosphate buffer were added to the pellet of algal cells after centrifugation under nitrogen atmosphere, and the suspension was incubated for a specified reaction time (20 to 30 min) in the dark. The hydrogenase activity was measured as the rate of hydrogen evolution from reduced methyl viologen. Light intensity distribution The distribution of light intensity within the bubble column was measured with a diffusing sphere photoprobe as described previously (3, 15) , which was made of a polymethylmethacrylate resin sphere 3.2 mm in diameter and plastic fiber optics.
RESULTS Effect of shading on the rate of hydrogen photoevolution The rates of hydrogen photoevolution are shown in Fig. 1. At high cell concentrations, the second peak of hydrogen evolution was observed 20 h after the start of illumination, the width of the second peak being much broader than the initial peak (Fig. 1A). When the column wall was not covered with a sheet of film impervious to light (Fig. 1A, B, open circles), a distinct initial gush of hydrogen was observed shortly after the start of illumination, while the initial peak of hydrogen was low or completely disappeared when a part of the column was covered (Fig. 1B, C, solid circles). The most noticeable effect of the film was that, except the initial peaks, the rate of photoevolution of hydrogen was higher in the cases with partial shading than without shading, the effect being more noticeable under the conditions of lower cell concentrations. Figure 2 shows the hydrogen evolution rate after the initial peak, i.e., that at the steady state or the top of the second peak. The lowest VL shown in Fig. 2, 0.17, means that the whole area of the wall of the glass column except the conical bottom was covered with the film. The rates of hydrogen photoevolution were higher at lower ratios of lighted volume VL. This dependence of hydrogen photoevolution rate on the lighted volume is contrary to that in normal photochemical reactions, where the overall reaction rates increase with the lighted volume ratio. Chlorophyll a fluorescence induction curves Figure 3 shows the variation with time in the chlorophyll a fluorescence induction curves measured after 5 min dark adaptation, which correspond to the course of H2 production shown in of Fig. 1A. While the rates of hydrogen photoevolution were sustained at a high level, the fluorescence induction curves showed characteristic variations corresponding to the phases named I, D, P, and S (14, 16, 17). The I-peak is the highest point in the induction curve, which is the same phenomenon reported for Scenedesmus cells kept under dark and anaerobic conditions, and the I-peak is caused by inactivation of photosystems in the dark and their reactiva-
FIG. 1. Hydrogen photoevolution rate and hydrogenase activity with and without partial shading. Algal cell concentrations: (A) 1.3 and 1.4 kg/m3; (B) 0.69 and 0.75 kg/m3; (C) 0.45 and 0.46 kg/m3. Values of VL in hydrogenase activity data: open squares, 1.0; solid squares, 0.17.
FIG. 2. Hydrogen photoevolution rate as a function of algal cell concentration.
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FIG. 5. Total hydrogen volume produced as a function of algal cell concentration. FIG. 3. Fluorescence induction curves in the course of hydrogen photoevolution shown in Fig. 1A. The figures at the top of the graph indicate the time at which each algal sample was obtained. Values of VL: open circles, 1.0; solid circles, 0.17.
FIG. 6. Cross-sectional distribution of light intensity within the bubble column photobioreactors filled with algal cell suspension. FIG. 4. Effect of lighted volume ratio (VL) and dark adaptation periods (t) on fluorescence induction curves during the steady hydrogen photoevolution. Algal cell concentration: 0.7 kg/m3. (A) VL = 1.0 [-]; (B) VL = 0.47 [-]; (C) VL = 0.17 [-].
tion by actinic light (17–19). The P-peak, which was observed about 5 s after the illumination of the actinic light, reflects the reactivation of inactivated ferredoxin-NADP+ reductase by the actinic light and the consequent resumption of electron flow through the Calvin cycle (20, 21). In Fig. 3, the heights of the I- and P-peaks decreased as the rate of hydrogen evolution gradually decreased, indicating that the mediating electron carriers were gradually damaged, and that the partial shading serves to preserve the initial shape of fluorescence induction curves, i.e., the initial photosynthetic activity is maintained over a longer period by partial shading. Figure 4 shows the fluorescence induction curves during the steady phase of H2 photoevolution under experimental conditions similar to that in Fig. 1B. The induction curves were obtained for three different VL values and for two different dark adaptation periods after sampling from the photobioreactor (t = 30 s and 10 min). The induction curves for
t = 10 min are similar to those in Fig. 3, while the heights of the I- and P-peaks for t = 30 s are lower than those for t = 10 min. These characteristics will be discussed later. Total hydrogen volume Figure 5 shows the total hydrogen volume photoproduced as functions of algal cell concentration. Although the total volume of hydrogen increased with decreasing ratio of the lighted area, the difference in the total volume with and without the films decreased as the cell concentration increased. The higher total hydrogen volume in cases with partial shading is due to the combined effects of the increase in the hydrogen evolution rate and the preservation of the photosystems including the activity of hydrogenase. The maximum total hydrogen volume for VL = 0.17 was 6.9 ´10–2 m3/kg at the cell concentration of 0.6 kg/m3, which is 1.5 times higher than for the cases without partial shading. Light intensity distribution within the culture vessel Figure 6 shows cross-sectional distributions of light intensity within the bubble column filled with algal suspension. The values shown in Fig. 6 represent the relative light intensity expressed as percentages of the reference intensity (6500 lx), which was measured at the inside wall of the
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FIG. 7. Light intensity distribution on the axis of bubble column photobioreactors with and without partial shading. Algal cell concentrations: (A) 0.26 kg/m3; (B) 0.73 kg/m3; (C) 0.96 kg/m3; (D) 1.29 kg/m3.
vessel filled with distilled water. The relative light intensity distributions on the axis of the column are shown in Fig. 7, where the regions of the depth larger than 14 cm with high relative light intensity correspond to the conical part of the bubble column bioreactor. On the basis of the cross-sectional and axial light intensity distribution, the volume ratio of the lighter region (to the total culture volume), where the relative light intensity is higher than 50% (3250 lx) or 28% (1820 lx) of the reference intensity, was calculated (Fig. 8). For instance, at a cell concentration of 1 g/l, the volume lighter than 1820 lx is 45% of the total suspension volume at VL = 1.0 (Fig. 8A), while it is only 7% at VL = 0.17 (Fig. 8C). The results in Fig. 8 as well as in Fig. 2 lead to the conclusion that the highest rate of hydrogen photoevolution was obtained when a large part (more than 90%) of the algal suspension was kept under low illumination of darker than 1800 lx. DISCUSSION The rate of hydrogen photoevolution increased as the volume ratio of the lighted region decreased (Fig. 2). This relationship between the area of the lighted region and the reaction rate is contrary to normal photochemical reactions. A possible explanation for this phenomenon is that the partitioning of electrons between the hydrogen photoevolution pathway via hydrogenase and the Calvin cycle (6, 7) is involved in the following manner. As described in Fig. 3, the I- and P-peaks reflect the rapid reactivation of photosystems and the regulation of electron flow via the ferredoxin-
FIG. 8. Volume ratio of lighter region (to the total culture volume) where the relative light intensity is higher than 50% or 28% of reference intensity (6500 lx). (A) VL = 1.0 [-]; (B) VL = 0.47 [-]; (C) VL = 0.17 [-].
NADP+ reductase (FNR), respectively, both by actinic light. In Fig. 4, the fluorescence induction curves obtained for t = 10 min show characteristic peaks of I and P of dark-adapted algal cells (Figs. 4A-2, B-2, C-2). On the other hand, the shapes of the induction curves for t = 30 s are determined by the illumination conditions for the bioreactors, i.e., the curves correspond to photosynthetic activities of the cells adapted to different light conditions within the reactors. When the lighted volume ratio is low (Fig. 4C-1), distinct I- and Ppeaks are observed as in the case of t = 10 min, indicating that dark-adapted cells are predominant in the photobioreactor. On the other hand, both peaks are less distinct at higher lighted volume ratios, corresponding to higher proportions of light-adapted cells with reactivated photosystems (Fig. 4A-1, B-1). On the basis of these observations, the inverse relation between the lighted volume ratio and hydrogen photoevolution rate can be explained as follows. If a part of the column wall is covered with the film impervious to light, the photosystems and FNR are inactivated while they are carried into the dark region by the liquid flow. When these dark-adapted cells are carried into the lighted region again, electron flow via the ferredoxin is partitioned mainly to light-driven hydrogen evolution during the lag time (about 5 s) before FNR is reactivated (6, 21). Although the electrons are supplied also to the Calvin cycle after the
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reactivation of the FNR, and consequently the rate of hydrogen photoevolution could be reduced, if the ratio of the lighted region is properly low, the algal cells with high hydrogen production rate will be predominant in the lighted region, and the overall light-driven hydrogen production rate could be kept at a higher value for the columns with partial shading. The effects of active oxygens on H2 photoevolution are discussed from the viewpoint of damage to photosystems or hydrogenase. As described in Fig. 3, damage to photosystems is not apparent while the H2 photoevolution rates were sustained at a high level, which indicates that the lower hydrogen evolution rates observed in the higher VL conditions are not due to the damage in photosystems. As for damage to hydrogenase, the hydrogenase activity remained relatively stable even after cessation of H2 photoevolution (Fig. 1C). If it is assumed that the rate of H2 photoevolution is proportional to the product of the hydrogenase activity and VL value, at 23 h after the start of illumination, as shown in Fig. 1C, the ratio of H2 evolution rate with partial shading to that without shading would be approximately (4.7 ´10–7 ´0.17)/ (2.7 ´10–7 ´1.0) = 0.3. The actual ratio at the same time point is (1.3´10–10)/(0.48´10–10)=2.7. This indicates that although the hydrogenase activity might be partly inhibited by active oxygens, the high values of H2 evolution rate for the partial shading conditions could be explained by the kinetic behavior of FNR, i.e., decrease in the proportion of electrons partitioned to the Calvin cycle prior to the reactivation of FNR in the light. Finally, the effect of light activation of the Calvin cycle enzymes on H2 photoevolution is briefly discussed. While electrons from photosystem I are transferred via ferredoxin to the light-regulated FNR as reducing power for the synthesis of carbohydrates, a part of the electrons from ferredoxin is transmitted to ferredoxin–thioredoxin reductase (FTR) as a regulatory electron signal to activate the Calvin cycle enzymes in the light (22, 23). In the above discussion, the regulation of hydrogen photoevolution rate is assumed to result from the linear electron transport via FNR and not from the electron signal via FTR. In conclusion, this study demonstrates that the photobioreactors with proper partial shading are better suited for hydrogen production in that the evolution rate is higher and the photosystems or necessary enzymes are better preserved. REFERENCES 1. Pow, T. and Krasna, A. I.: Photoproduction of hydrogen from water in hydrogenase-containing algae. Arch. Biochem. Biophys., 194, 413–421 (1979). 2. Rosenkrans, M. and Krasna, A. I.: Stimulation of hydrogen photoproduction in algae by removal of oxygen by reagents that combine reversibly with oxygen. Biotechnol. Bioeng., 26, 1334–1342 (1984). 3. Kojima, E. and Yamaguchi, Y.: Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa. J. Ferment. Technol., 66, 19–25 (1988). 4. Kojima, E.: Effect of inorganic salts on the duration of hydrogen photoevolution by Chlorella. Kagaku Kogaku Ronbun-
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