Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.     

An outdoor biophotolytic system using the cyanobacterium Anabaena cylindrica B629

The cyanobacterium Anabaena cylindrica B629 was suspended in small glass beds and incubated in a gas-tight glass vessel outdoors under a gas atmosphere comprising carbon monoxide (0.2%), acetylene (5%), oxygen (6.5%), and nitrogen. The solution phase initially contained sodium bicarbonate (10mM) at pH 7. Under these conditions the organism continuously produced hydrogen gas for over three weeks. The temperature of the culture was maintained below 30°C and minimum night temperatures were recorded. The vessel was covered by a shadecloth, which reduced the natural illumination by approximately 70%. The system is an alternative to those requiring the strict absence of oxygen and little nitrogen, and requires virtually no attention during the incubation period.     

Comparative Amperometric Study of Uptake Hydrogenase and Hydrogen Photoproduction Activities between Heterocystous Cyanobacterium Anabaena cylindrica B629 and Nonheterocystous Cyanobacterium Oscillatoria sp. Strain Miami BG7

Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N₂ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production, photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O₂ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O₂ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7.     

Anaerobic and Aerobic Hydrogen Gas Formation by the Blue-Green Alga Anabaena cylindrica

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5′-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Nitrogen fixation by Anabaena cylindrica

Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H₂ (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H₂ and was strongly inhibited by oxygen (50% inhibition at about 5–6% O₂). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O₂ on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO₂ fixation by vegetative cells.     

Emerson enhancement of carbon fixation but not of acetylene reduction (nitrogenase activity) in Anabaena cylindrica

Summary Enhancement of carbon fixation was demonstrated in the bluegreen alga, Anabaena cylindrica, grown in either aerobic or microaerobic conditions. Under identical conditions no enhancement of acetylene reduction was observed. Light absorbed by photosystem I supported relatively more acetylene reduction than carbon fixation. No competition between the two processes was observed under light-limiting conditions. The findings suggest that carbon fixation and acetylene reduction may depend on different pools of reductant and ATP. When aerobically grown cells were placed in the dark or at limiting light intensities, acetylene reduction was higher in air than under argon. In contrast, carbon fixation was lower in air than in argon.     

Anaerobic and aerobic hydrogen gas formation by the blue-green alga Anabaena cylindrica.

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5'-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Hydrogenase activity in the thermophile Mastigocladus laminosus

Hydrogenase activity in the thermophilic cyanobacterium, $\text{Mastigocladus}$$\text{laminosus}$ was studied both $\text{in vivo}$ and $\text{in vivo}$ hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H₂-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O₂-independent reaction. $\text{In vitro}$ hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for $\text{Anabaena cylindrica}$.     

Hydrogen metabolism by filamentous cyanobacteria

Apparent discrepancies in the literature concerning the amounts of H₂ produced by strains of Anabaena cylindrica are explained. These are not due to differences in strains used by different workers nor to differences in growth conditions, but rather appear to be due to the fact that cultures show an increasing dependence with age on CO₂ for sustained H₂ production. Two distinct hydrogenase activities were measured and characterized, both in vivo and in vitro in A. cylindrica B629; these were H₂ uptake activity and H₂ evolution from reduced methyl viologen. Gentle cell disruption techniques were used to gain further evidence that the latter activity was soluble. H₂ uptake was strongly inhibited by acetylene in vivo in the light or in the dark with phenazine methosulfate added, but only after a prolonged lag period. In extracts this lag did not occur. A detailed study of the nitrogenase and hydrogen uptake activities and their interrelationship both in the light and in the dark in A. cylindrica B629 showed that only in the dark in the presence of O₂ did H₂ uptake support C₂H₂ reduction significantly. Under several conditions in which nitrogenase activity was inhibited H₂ uptake was unaffected. H₂ metabolism was tested in three nonheterocystous filamentous cyanobacteria under different growth and incubation conditions. These were Plectonema boryanum, Schizothrix calcicola, and Oscillatoria brevis. Myxosarcina chroococcoides and Fischerella muscicola were also investigated. Cyanobacterial species vary markedly in their hydrogen metabolism and in the composition of the three H₂ metabolizing enzymes.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca 1 b

Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 μmoles H₂/min·mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5–10 μM concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH₂. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the K _m was 0.063 mM H₂ and for saturating hydrogen concentration the K _m was 0.123 mM NAD.     

Amperometric responses to acetylene, ethylene, and methane by a Clark-type oxygen electrode

Hydrogen-conditioned Clark electrodes exposed to successive acetylene samples displayed a decreasing curvilinear response. Slight electrode responses are also effected by saturated ethylene solutions. Thus, amperometric measurements of hydrogen or carbon monoxide in the aqueous environment by these electrodes are perturbated by the presence of acetylene and to a much lesser extent by ethylene. Methane-saturated solutions induced no electrode response.     

Respiration and nitrogen fixation in nodulated roots of soya bean and pea

Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules.     

Energy coupling and respiration in Nitrosomonas europaea.

Intact cells of Nitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. The Km value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of "endogenous" substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.     

Hydrazine as a substrate and inhibitor of Azotobacter vinelandii nitrogenase

Hydrazine has been tested as a substrate and inhibitor of nitrogenase from Azotobacter vinelandii. It is a linear noncompetitive inhibitor of acetylene reduction, with K_il = K_is = 80 mM at pH 8.0. Carbon monoxide is a linear noncompetitive inhibitor of hydrazine reduction with K_ii = K_is = 2 × 10^?4atm. The inhibition of acetylene reduction by hydrazine is unaffected by the presence of hydrogen, and hydrogen does not inhibit the reduction of hydrazine. Hydrazine can completely suppress hydrogen evolution, while not inhibiting phosphate hydrolysis. The apparent K_m for hydrazine reduction varies with pH, reaching a limiting value of about 25 mM at high pH. The apparent K_i for hydrazine inhibition of hydrogen evolution reaches a similar limiting value at high pH. By varying the concentration of ATP it is possible to alter the relative allocation of electrons to acetylene or hydrazine. Hydrazine is a relatively more potent inhibitor of acetylene reduction at low levels of ATP. It is concluded that hydrazine is able to react effectively with a less reduced state of the enzyme from A. vinelandii than is acetylene or dinitrogen.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.     

Photoproduction of Hydrogen by the Marine Heterocystous Cyanobacterium Anabaena Species TU37-1 Under a Nitrogen Atmosphere

Hydrogen production rates by Anabaena sp. strain TU37-1 obtained after an initial 1-day incubation period were approximately 70 to 80 and 3 to 9 µmol (mg chl)^–1 h^–1 under argon and nitrogen atmospheres, respectively. Hydrogen production under argon was not enhanced by addition of carbon dioxide, but was enhanced to some extent under nitrogen by increasing the initial carbon dioxide concentration. Rates of hydrogen and oxygen production during the initial 7-hour period were 15 and 220 µmol (mg chl)^–1 h^–1, respectively, in vessels with 18.5% initial carbon dioxide. Hydrogen production under nitrogen was enhanced by addition of carbon monoxide (1%). The rate obtained from the initial 1-day incubation period was about 40 µmol (mg chl)^–1 h^–1, which corresponded to about 60% of that under argon. On the basis of these observations, a possible strategy for hydrogen production by nitrogen-fixing cyanobacteria under nitrogen in the presence of carbon monoxide is indicated.     

Formation of proteinoid microspheres under simulated prebiotic atmospheres and individual gases.

The formation of microspheres from acidic and basic proteinoids was attempted under simulated prebiotic atmospheres and constituent gases thereof. Both types of proteinoid yielded microspheres under carbon dioxide, carbon monoxide, methane, hydrogen sulfide, hydrogen, nitrogen, and oxygen (tested separately) and also under nitrogen-carbon dioxide atmospheres; higher proportions of carbon dioxide resulted in fewer spheres from basic proteinoid. Neither type of proteinoid formed spheres on 10-minute exposure to ammonia or methane-hydrogen-ammonia atmospheres. (Brief exposure resulted in spheres from basic proteinoid.) The effects, both qualitative and quantitative, were indicated by control experiments to be due to pH, rather than to the specific gas (or ion). The results suggest that the proteinoid microsphere model for protocells is applicable under a variety of possible prebiotic atmospheres, with some restrictions imposed by pH.     

Gas Exchange of Algae: II. Effects of Oxygen, Helium, and Argon on the Photosynthesis of Chlorella pyrenoidosa

Changes in the oxygen partial pressure of air over the range of 8 to 258 mm of Hg did not adversely affect the photosynthetic capacity of Chlorella pyrenoidosa. Gas exchange and growth measurements remained constant for 3-week periods and were similar to air controls (oxygen pressure of 160 mm of Hg). Oxygen partial pressures of 532 and 745 mm of Hg had an adverse effect on algal metabolism. Carbon dioxide consumption was 24% lower in the gas mixture containing oxygen at a pressure 532 mm of Hg than in the air control, and the growth rate was slightly reduced. Oxygen at a partial pressure of 745 mm of Hg decreased the photosynthetic rate 39% and the growth rate 37% over the corresponding rates in air. The lowered metabolic rates remained constant during 14 days of measurements, and the effect was reversible after this time. Substitution of helium or argon for the nitrogen in air had no effect on oxygen production, carbon dioxide consumption, or growth rate for 3-week periods. All measurements were made at a total pressure of 760 mm of Hg, and all gas mixtures were enriched with 2% carbon dioxide. Thus, the physiological functioning and reliability of a photosynthetic gas exchanger should not be adversely affected by: (i) oxygen partial pressures ranging from 8 to 258 mm of Hg; (ii) the use of pure oxygen at reduced total pressure (155 to 258 mm of Hg) unless pressure per se affects photosynthesis, or (iii) the inclusion of helium or argon in the gas environment (up to a partial pressure of 595 mm of Hg).