APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1978, p. 704-710 0099-2240/78/0035-0704$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 35, No. 4
Printed in U.S.A.
Hydrogen Production by Anabaena cylindrica: Effects of Varying Ammonium and Ferric Ions, pH, and Light THOMAS W. JEFFRIES,t HECTOR TIMOURIAN,* AND RAYMOND L. WARD Biomedical Sciences Division and General Chemistry Division, Lawrence Livernore Laboratory, University of California, Livermore, California 94550 Received for publication 10 November 1977
Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 ,ul of H2 per mg [dry wt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3" per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation. The production of hydrogen from water by using a biological catalyst and sunlight as an energy source (biophotolysis) could substitute for natural gas (11). However, to establish a technically and economically viable system, the biological catalyst must be synthesized inexpensively, operate efficiently, and be stable for at least several weeks. According to various proposed models (20, 22, 24), the vegetative cells of heterocystous, filamentous, blue-green algae fix C02 by using both photosystem I and photosystem II to split water and generate reducing agent plus O2. The reducing agent is then transferred to the heterocyst. Nitrogenase in the heterocyst utilizes the reducing agent plus adenosine 5'-triphosphate, which is produced by photo- and oxidative phosphorylation, to reduce N2 to NH3. In the absence of N2, electrons are transferred to hydrogen ions to form H2. Photosystem II is not found in the heterocyst (7, 18, 19), and the heterocysts have a reducing intracellular environment (14) so that the oxygen-labile nitrogenase is maintained under optimal conditions for maximal activity. A biophotolysis system using intact, heterocystous, filamentous blue-green algae (3,12) possesses several unique characteristics, compared with green algae or cell-free hydrogen systems. Compared with systems using green algae (5, 8, 9, 17), biophotolysis by heterocystous blue-green algae is inhibited less by the coproduction of 02 (15, 21, 22) and, hence, would be more active at t Present address: Department of Chemical Engineering and Applied Chemistry, Columbia University, New York, NY 10027.
elevated concentrations of H2 and 02. Although cell-free systems (2, 13) might eventually attain a higher efficiency of light conversion than intact cells under laboratory conditions (10), the cost and stability would limit their economic exploitation (4). Intact cells of heterocystous, filamentous bluegreen algae have produced H2 continuously for 7 to 19 days with an efficiency of 0.4% when grown in a nitrogen-free medium and incubated under an argon atmosphere (22). After 2 to 3 days however, the algal filaments tended to break up and H2 production declined unless fixed nitrogen was supplied to the cells. The periodic addition of 0.1 mM NH4Cl did not inhibit H2 evolution; indeed, cultures without nitrogen additions were found to lose activity after 1 day of starvation (22). Various inherent advantages are offered by an intact-cell biophotolysis system that is capable of self-protection from coproduced O2. Therefore, the filamentous blue-green algae are obvious candidates for further development. Our objectives in this research were to determine optimal nutritional and physiological conditions necessary for continuous, stable H2 production by filamentous blue-green algae by manipulating light intensity, nitrogen addition, and pH. MATERIALS AND METHODS Organisms and culture methods. Anabaena cylindrica (Smith) was obtained from J. Smith (Australian National University, Canberra, Australia). A. cylindrica UTCC 629 (Fogg) was obtained from J. R. Benemann (University of California, Berkeley, Calif.). 704
VOL. 35, 1978
HYDROGEN PRODUCTION BY ANABAENA CYLINDRICA
Cells were routinely cultivated in the medium of Allen and Stanier (1), which was modified by the substitution of ferric citrate and citric acid with 5.0 mg of Fe3" per liter as an ethylenediamenetetraacetic acid complex. Cultures streaked for purity on agar plates were massively inoculated into 250 ml of liquid medium in a 500-ml Erlenmeyer flask and incubated at 27°C under 23 W of fluorescent light per m2 on a tabletop shaker (125 rpm). After 7 to 10 days cells were centrifuged and aseptically transferred either to 3,500 ml of fresh medium in a clear, glass, 1-gallon (about 3.79 liters) jug or to 800 ml of medium in a 900-ml Roux bottle. The Roux bottles were modified by turning the neck 90% to the vertical. This allowed the bottles to lie horizontally in tungsten-illuminated cabinets. Cultures were grown at 27°C under 32 W of light per m2 and sparged with N2-0.5% CO2. All cultures were agitated with magnetic stirring bars. Cell mass was estimated with a Klett-Summerson photoelectric colorimeter (model 800-3) fitted with a Beckman 500-nm filter. Neither chlorophylls nor phycocyanin absorb significantly at this wavelength, so the optical density correlates well with dry-weight standard curves even after phycocyanin loss during nitrogen starvation. Within the linear region (less than 300 Klett units), 1 Klett unit was equivalent to either 0.52 (A. cylindrica, Smith) or 0.87 (A. cylindrica, Fogg) yg (dry wt)/ml of culture suspension. Cultures grown in 1-gallon jugs were divided into sterile Roux bottles. When cultures reach 0.075 to 0.5 mg (dry wt)/ml, argon sparging was initiated (t = 0). Replicate cultures were sparged with 5 to 15 ml of argon-CO2 per min. Gas flow rates to the different cultures were regulated by passage through a fourchannel Buchler polystatic pump. Carbon dioxide was mixed with the argon in varying proportions so that the pH of the cultures could be controlled between 6.5 and 9.5. Illumination. Cultures were incubated in cabinets with either fluorescent or tungsten light. The fluorescent light cabinet consisted of two parallel, opposed banks (1.22 by 0.5 m; eight lamps per bank) of 40-W "naturescent" lamps (Duro-Lite Lamps, Inc., Fairlawn, N.J.). According to the manufacturer's specifications, these lamps exhibit spectral characteristics that are very close to those of the visible portion of normal sunlight. The cultures in 900-ml Roux bottles were placed in the center of the cabinet, 16 cm from either bank of lights. Light intensity was measured with a silicon photodiode (United Detector Technology Inc., Santa Monica, Calif.; model TIN-1ODF). This instrument measures all incident light from a 180° hemisphere as corrected for cosine losses. Average light intensity falling on all surfaces of the Roux bottle, as measured by this method, was 32 W/m2. The total estimated surface area of the flask was 625 cm2, so the total incident light on each flask was 2.0 W. These figures did not take into account light lost by reflection from the glass. Light passing through the culture was measured during various experiments but was not subtracted when calculating the efficiency of hydrogen production. These values ranged from close to 0 to almost 15% of the total incident light. The tungsten light cabinets were modified glass-top Beckman mobile refrigeration units (model 133A).
705
Tungsten reflector lamps were mounted externally to provide mumination from the top. Cultures in modified 900-ml Roux bottles were incubated horizontally, about 30 cm from the light sources. Light intensity was varied by raising or lowering the cultures or by using bulbs of different wattages. Constant uniform temperature (27°C) was maintained by a combination of the thermostated refrigeration unit and air circulation fans. Light intensity on the cultures was measured during each e*xperiment. In the tungsten cabinet, the light intensity was 6 W/m2. The calculated surface area of one side of the modified Roux flask (assuming the flask was transected by a horizontal plane) was 215 cm2, so that the total incident light was 0.127 W. The efficiency of photosynthetic conversion of light to H2 was calculated as the free energy of the total amount of H2 produced divided by the total energy of the light incident on the culture vessels times 100. The free energy available from the combustion of H2 to form water is 217.0 kJ/mol. Hydrogen production and acetylene reduction assays. Hydrogen production was measured in two ways: sampling from a continuously sparged culture or assaying total production for a period of time in a sealed vial. For cultures that were sparged continuously with argon, 20-gauge needles were connected to the effluent tubes, and rubber-stoppered, 25-ml serum vials were then perforated by the effluent needle and vented with a second needle. Effluent gas passed continuously through the sampling vials. The total gas volume over the surface of the culture, including the sampling vial, was less than 60 ml. With an argon flow rate of 5 to 15 ml/min, one-half of the gas over the culture was replaced every 4 to 12 min. Sampling vials were periodically removed and replaced to obtain sequential gas samples. Thus, effluent gas samples could be obtained without disturbing the cultures. Gas bubbles tended to accumulate near the tops of the cultures along with clumps of cells. These gases were released when the cell masses were broken up by agitation, but agitation could cause large fluctuations in the measured hydrogen; therefore, samples were not taken within 6 h of any disturbance of the culture vessel. Total hydrogen production and acetylene reduction were also assayed in either 20- or 25-ml serum vials under the same light intensities as the parent cultures. Cell suspensions (3.0 ml) were placed in each vial and flushed with at least an equivalent of 30 changes in volume (750 ml) of argon. For H2 production assays, vials were placed directly in the light after sparging. For acetylene reduction (nitrogenase) assays, vials were injected with 2.0 ml of acetylene, vented to the atmosphere, and sealed with plastic tape. All assays were performed in triplicate. Samples were routinely incubated for 6 h. Reactions were stopped by the injection of 1.5 ml of 10% trichloroacetic acid. In the text these are referred to as "stop-time assays" to distinguish them from the sampling of the continuous cultures. Hydrogen samples were analyzed chromatographically with a thermister detector and a 5-A molecularsieve column (Supelco, Inc., Bellefonte, Pa.). Acetylene and ethylene were analyzed with a flame ionization detector on a Porapak R column (Waters Associates, Inc., Framingham, Mass.). Data were electron-
JEFFRIES, TIMOURIAN, AND WARD
706
ically integrated, and concentrations were determined from mixtures of standard gases. For determination of the frequency of heterocysts, a minimum of 50 heterocysts (an average of 1,000 vegetative cells) were counted at x 100 magnification, using a phase microscope. A cell was termed a proheterocyst if cytoplasmic differentiation had been initiated and if plug material was not apparent.
RESULTS In general, hydrogen production could be measured in cultures of A. cylindrica starting at 3 days after incubation in an argon atmosphere. The rate of hydrogen production increased rapidly, reaching a maximum after 6 to 9 days of incubation. At this time, the culture had become yellow, due to depletion of phycocyanin. The rate of hydrogen production then started to decrease (Fig. 1A). We describe here experimental manipulations aimed at extending hydrogen production. The times of nitrogen additions and many ofthe manipulations were empirically chosen. The main aim was to prevent nitrogen starvation from inactivating the cells. As nitrogen starvation proceeded, the cultures turned yellow and the filaments started to break. Effects of ammonium chloride and me30I -j u
c b.
220
/
1
10
-
0
(
thionine sulfoximine. To examine the effects of nitrogen addition on H2 production, NH4Cl was added either alone or in the presence of methionine sulfoximine to cultures of A. cylindrica. Methionine sulfoximine is known to release inhibition of the vegetative cell's heterocyst-forming ability by interaction with glutamine synthetase (16). Figure 1 illustrates the results of an experiment with four simultaneous cultures of A. cylindrica (Fogg). For the most part, in all cultures H2 production and acetylene-to-ethylene reduction closely followed each other as a function of time. Culture A, without any added ammonium chloride, produced H2 and ethylene for the longest continuous period (18 days). Hydrogen was produced for the shortest period (6 days) in the culture that received ammonium chloride every 2 days (culture C). Delaying the time of the first ammonium chloride addition (culture B) or adding methionine sulfoximine (culture D) increased the length of time that H2 was formed to 12 and 9 days, respectively. Maximum rates of hydrogen production were attained after 6 days of argon sparging. The transition from low to high rates of H2 production was accompanied by a change in the ap-
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APPL. ENVIRON. MICROBIOL.
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Vol. 35, No. 4
Printed in U.S.A.
Hydrogen Production by Anabaena cylindrica: Effects of Varying Ammonium and Ferric Ions, pH, and Light THOMAS W. JEFFRIES,t HECTOR TIMOURIAN,* AND RAYMOND L. WARD Biomedical Sciences Division and General Chemistry Division, Lawrence Livernore Laboratory, University of California, Livermore, California 94550 Received for publication 10 November 1977
Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 ,ul of H2 per mg [dry wt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3" per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation. The production of hydrogen from water by using a biological catalyst and sunlight as an energy source (biophotolysis) could substitute for natural gas (11). However, to establish a technically and economically viable system, the biological catalyst must be synthesized inexpensively, operate efficiently, and be stable for at least several weeks. According to various proposed models (20, 22, 24), the vegetative cells of heterocystous, filamentous, blue-green algae fix C02 by using both photosystem I and photosystem II to split water and generate reducing agent plus O2. The reducing agent is then transferred to the heterocyst. Nitrogenase in the heterocyst utilizes the reducing agent plus adenosine 5'-triphosphate, which is produced by photo- and oxidative phosphorylation, to reduce N2 to NH3. In the absence of N2, electrons are transferred to hydrogen ions to form H2. Photosystem II is not found in the heterocyst (7, 18, 19), and the heterocysts have a reducing intracellular environment (14) so that the oxygen-labile nitrogenase is maintained under optimal conditions for maximal activity. A biophotolysis system using intact, heterocystous, filamentous blue-green algae (3,12) possesses several unique characteristics, compared with green algae or cell-free hydrogen systems. Compared with systems using green algae (5, 8, 9, 17), biophotolysis by heterocystous blue-green algae is inhibited less by the coproduction of 02 (15, 21, 22) and, hence, would be more active at t Present address: Department of Chemical Engineering and Applied Chemistry, Columbia University, New York, NY 10027.
elevated concentrations of H2 and 02. Although cell-free systems (2, 13) might eventually attain a higher efficiency of light conversion than intact cells under laboratory conditions (10), the cost and stability would limit their economic exploitation (4). Intact cells of heterocystous, filamentous bluegreen algae have produced H2 continuously for 7 to 19 days with an efficiency of 0.4% when grown in a nitrogen-free medium and incubated under an argon atmosphere (22). After 2 to 3 days however, the algal filaments tended to break up and H2 production declined unless fixed nitrogen was supplied to the cells. The periodic addition of 0.1 mM NH4Cl did not inhibit H2 evolution; indeed, cultures without nitrogen additions were found to lose activity after 1 day of starvation (22). Various inherent advantages are offered by an intact-cell biophotolysis system that is capable of self-protection from coproduced O2. Therefore, the filamentous blue-green algae are obvious candidates for further development. Our objectives in this research were to determine optimal nutritional and physiological conditions necessary for continuous, stable H2 production by filamentous blue-green algae by manipulating light intensity, nitrogen addition, and pH. MATERIALS AND METHODS Organisms and culture methods. Anabaena cylindrica (Smith) was obtained from J. Smith (Australian National University, Canberra, Australia). A. cylindrica UTCC 629 (Fogg) was obtained from J. R. Benemann (University of California, Berkeley, Calif.). 704
VOL. 35, 1978
HYDROGEN PRODUCTION BY ANABAENA CYLINDRICA
Cells were routinely cultivated in the medium of Allen and Stanier (1), which was modified by the substitution of ferric citrate and citric acid with 5.0 mg of Fe3" per liter as an ethylenediamenetetraacetic acid complex. Cultures streaked for purity on agar plates were massively inoculated into 250 ml of liquid medium in a 500-ml Erlenmeyer flask and incubated at 27°C under 23 W of fluorescent light per m2 on a tabletop shaker (125 rpm). After 7 to 10 days cells were centrifuged and aseptically transferred either to 3,500 ml of fresh medium in a clear, glass, 1-gallon (about 3.79 liters) jug or to 800 ml of medium in a 900-ml Roux bottle. The Roux bottles were modified by turning the neck 90% to the vertical. This allowed the bottles to lie horizontally in tungsten-illuminated cabinets. Cultures were grown at 27°C under 32 W of light per m2 and sparged with N2-0.5% CO2. All cultures were agitated with magnetic stirring bars. Cell mass was estimated with a Klett-Summerson photoelectric colorimeter (model 800-3) fitted with a Beckman 500-nm filter. Neither chlorophylls nor phycocyanin absorb significantly at this wavelength, so the optical density correlates well with dry-weight standard curves even after phycocyanin loss during nitrogen starvation. Within the linear region (less than 300 Klett units), 1 Klett unit was equivalent to either 0.52 (A. cylindrica, Smith) or 0.87 (A. cylindrica, Fogg) yg (dry wt)/ml of culture suspension. Cultures grown in 1-gallon jugs were divided into sterile Roux bottles. When cultures reach 0.075 to 0.5 mg (dry wt)/ml, argon sparging was initiated (t = 0). Replicate cultures were sparged with 5 to 15 ml of argon-CO2 per min. Gas flow rates to the different cultures were regulated by passage through a fourchannel Buchler polystatic pump. Carbon dioxide was mixed with the argon in varying proportions so that the pH of the cultures could be controlled between 6.5 and 9.5. Illumination. Cultures were incubated in cabinets with either fluorescent or tungsten light. The fluorescent light cabinet consisted of two parallel, opposed banks (1.22 by 0.5 m; eight lamps per bank) of 40-W "naturescent" lamps (Duro-Lite Lamps, Inc., Fairlawn, N.J.). According to the manufacturer's specifications, these lamps exhibit spectral characteristics that are very close to those of the visible portion of normal sunlight. The cultures in 900-ml Roux bottles were placed in the center of the cabinet, 16 cm from either bank of lights. Light intensity was measured with a silicon photodiode (United Detector Technology Inc., Santa Monica, Calif.; model TIN-1ODF). This instrument measures all incident light from a 180° hemisphere as corrected for cosine losses. Average light intensity falling on all surfaces of the Roux bottle, as measured by this method, was 32 W/m2. The total estimated surface area of the flask was 625 cm2, so the total incident light on each flask was 2.0 W. These figures did not take into account light lost by reflection from the glass. Light passing through the culture was measured during various experiments but was not subtracted when calculating the efficiency of hydrogen production. These values ranged from close to 0 to almost 15% of the total incident light. The tungsten light cabinets were modified glass-top Beckman mobile refrigeration units (model 133A).
705
Tungsten reflector lamps were mounted externally to provide mumination from the top. Cultures in modified 900-ml Roux bottles were incubated horizontally, about 30 cm from the light sources. Light intensity was varied by raising or lowering the cultures or by using bulbs of different wattages. Constant uniform temperature (27°C) was maintained by a combination of the thermostated refrigeration unit and air circulation fans. Light intensity on the cultures was measured during each e*xperiment. In the tungsten cabinet, the light intensity was 6 W/m2. The calculated surface area of one side of the modified Roux flask (assuming the flask was transected by a horizontal plane) was 215 cm2, so that the total incident light was 0.127 W. The efficiency of photosynthetic conversion of light to H2 was calculated as the free energy of the total amount of H2 produced divided by the total energy of the light incident on the culture vessels times 100. The free energy available from the combustion of H2 to form water is 217.0 kJ/mol. Hydrogen production and acetylene reduction assays. Hydrogen production was measured in two ways: sampling from a continuously sparged culture or assaying total production for a period of time in a sealed vial. For cultures that were sparged continuously with argon, 20-gauge needles were connected to the effluent tubes, and rubber-stoppered, 25-ml serum vials were then perforated by the effluent needle and vented with a second needle. Effluent gas passed continuously through the sampling vials. The total gas volume over the surface of the culture, including the sampling vial, was less than 60 ml. With an argon flow rate of 5 to 15 ml/min, one-half of the gas over the culture was replaced every 4 to 12 min. Sampling vials were periodically removed and replaced to obtain sequential gas samples. Thus, effluent gas samples could be obtained without disturbing the cultures. Gas bubbles tended to accumulate near the tops of the cultures along with clumps of cells. These gases were released when the cell masses were broken up by agitation, but agitation could cause large fluctuations in the measured hydrogen; therefore, samples were not taken within 6 h of any disturbance of the culture vessel. Total hydrogen production and acetylene reduction were also assayed in either 20- or 25-ml serum vials under the same light intensities as the parent cultures. Cell suspensions (3.0 ml) were placed in each vial and flushed with at least an equivalent of 30 changes in volume (750 ml) of argon. For H2 production assays, vials were placed directly in the light after sparging. For acetylene reduction (nitrogenase) assays, vials were injected with 2.0 ml of acetylene, vented to the atmosphere, and sealed with plastic tape. All assays were performed in triplicate. Samples were routinely incubated for 6 h. Reactions were stopped by the injection of 1.5 ml of 10% trichloroacetic acid. In the text these are referred to as "stop-time assays" to distinguish them from the sampling of the continuous cultures. Hydrogen samples were analyzed chromatographically with a thermister detector and a 5-A molecularsieve column (Supelco, Inc., Bellefonte, Pa.). Acetylene and ethylene were analyzed with a flame ionization detector on a Porapak R column (Waters Associates, Inc., Framingham, Mass.). Data were electron-
JEFFRIES, TIMOURIAN, AND WARD
706
ically integrated, and concentrations were determined from mixtures of standard gases. For determination of the frequency of heterocysts, a minimum of 50 heterocysts (an average of 1,000 vegetative cells) were counted at x 100 magnification, using a phase microscope. A cell was termed a proheterocyst if cytoplasmic differentiation had been initiated and if plug material was not apparent.
RESULTS In general, hydrogen production could be measured in cultures of A. cylindrica starting at 3 days after incubation in an argon atmosphere. The rate of hydrogen production increased rapidly, reaching a maximum after 6 to 9 days of incubation. At this time, the culture had become yellow, due to depletion of phycocyanin. The rate of hydrogen production then started to decrease (Fig. 1A). We describe here experimental manipulations aimed at extending hydrogen production. The times of nitrogen additions and many ofthe manipulations were empirically chosen. The main aim was to prevent nitrogen starvation from inactivating the cells. As nitrogen starvation proceeded, the cultures turned yellow and the filaments started to break. Effects of ammonium chloride and me30I -j u
c b.
220
/
1
10
-
0
(
thionine sulfoximine. To examine the effects of nitrogen addition on H2 production, NH4Cl was added either alone or in the presence of methionine sulfoximine to cultures of A. cylindrica. Methionine sulfoximine is known to release inhibition of the vegetative cell's heterocyst-forming ability by interaction with glutamine synthetase (16). Figure 1 illustrates the results of an experiment with four simultaneous cultures of A. cylindrica (Fogg). For the most part, in all cultures H2 production and acetylene-to-ethylene reduction closely followed each other as a function of time. Culture A, without any added ammonium chloride, produced H2 and ethylene for the longest continuous period (18 days). Hydrogen was produced for the shortest period (6 days) in the culture that received ammonium chloride every 2 days (culture C). Delaying the time of the first ammonium chloride addition (culture B) or adding methionine sulfoximine (culture D) increased the length of time that H2 was formed to 12 and 9 days, respectively. Maximum rates of hydrogen production were attained after 6 days of argon sparging. The transition from low to high rates of H2 production was accompanied by a change in the ap-
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