Stimulation of hydrogen photoproduction in algae by removal of oxygen by reagents that combine reversibly with oxygen

Hydrogen photoproduction from water by Scenedesmus cells was achieved in the presence of reagents that combine reversibly with oxygen. The oxygen can be subsequently released, and H(2) and O(2) are obtained in the 2:1 ratio expected for H(2)O photolysis. This was accomplished in an experimental design which facilitates rapid transfer of gases and the use of a variety of water-soluble and DMSO-soluble chelates of cobalt which combine reversibly with oxygen.     

The mechanism of hydrogen photoproduction by several algae

Summary The contribution of PS II to H₂ photoproduction by several unicellular green algae was measured both when O₂ evolution and photophosphorylation were unimpaired and also when these processes had been eliminated by Cl-CCP. As judged by the effects of DCMU, a PS II contribution was found under both sets of experimental conditions for several strains of Chlorella, Ankistrodesmus and Scenedesmus. However, H₂ photoproduction by Chlamydomonas moewusii was insensitive to DCMU and thus was entirely due to PS I. With cells treated with Cl-CCP, the relative amount of PS II contribution varied from zero in autotrophically grown Chlamydomonas reinhardii, to 20% in photoheterotrophically grown and 50% in autotrophically grown cells of Ankistrodesmus braunii, Chlorella fusca, Chlorella vulgaris and Scenedesmus obliquus. The dehydrogenation of reduced H-donors by PS II of Scenedesmus treated with Cl-CCP showed the same biphasic kinetics previously described for H₂ photoproduction by PS I of this alga.Abbreviations Cl-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ICC Indiana Culture Collection - PS photosystem - SAL salicylaldoxime - SIO Marine Botany Culture Collection, Scripps Institution of Oceanography These studies were supported by contract No. AT-(40-1)-2687 from the U.S. Atomic Energy Commission.     

Long-term endurance and selection studies in hydrogen and oxygen photoproduction by Chlamydomonas reinhardtii

Two wild-type strains of Chlamydomonas reinhardtii have been subjected to repeated cycles of anaerobiosis, carbon dioxide deprivation, and irradiation as a means of testing the long-term stability of hydrogen and oxygen photoproduction and the effectiveness of these conditions as selection or adaptation pressures for increasing hydrogen and/or oxygen yields. Simultaneous hydrogen and oxygen photoproduction yields were monitored in each culture for 160 h. The cells were then removed from the reaction chamber and used to inoculate fresh growth medium to produce the culture for the next experiment. This cycle was repeated five times. Yields of hydrogen and oxygen improved after three cycles and declined in the fourth and fifth; unlike the second and third cycles, extended periods of aerobic growth were used for the fourth and fifth cycles. The stability of hydrogen and oxygen photoproduction was greater in the fifth cycle than in any of the previous cycles. These subpopulations had hydrogen and oxygen production rates, at 160 h, which were nearly equal to the rates at the beginning of the fifth-cycle experiments. Time profiles of the relative hydrogen yields from each of the five cycles, prepared at 32, 80, and 120 h, show that the relative yield in each varies with the point in time at which the profile was taken. Chlorophyll retention increased with each successive cycle, indicating selection or adaptation for a more durable population of cells with respect to the light-harvesting component of the photosynthetic apparatus.     

Effect of oxygen dosing point and mixing on the microaerobic removal of hydrogen sulphide in sludge digesters

Limited oxygen supply to anaerobic sludge digesters to remove hydrogen sulphide from biogas was studied. Micro-oxygenation showed competitive performance to reduce considerably the additional equipment necessary to perform biogas desulphurization. Two pilot-plant digesters with an HRT of ∼20 d were micro-oxygenated at a rate of 0.25 NL per L of feed sludge with a removal efficiency higher than 98%. The way of mixing (sludge or biogas recirculation) and the point of oxygen supply (headspace or liquid phase) played an important role on hydrogen sulphide oxidation. While micro-oxygenation with sludge recirculation removed only hydrogen sulphide from the biogas, dissolved sulphide was removed if micro-oxygenation was performed with biogas recirculation. Dosage in the headspace resulted in a more stable operation. The result of the hydrogen sulphide oxidation was mostly elemental sulphur, partially accumulated in the headspace of the digester, where different sulphide-oxidising bacteria were found.     

Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems

A comparative study of H₂ photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H₂ by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H₂ photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H₂ was observed to occur with a rate of 350 to 400 micromoles H₂ per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O₂) is used to produce H₂.     

The kinetics of hydrogen photoproduction by adapted Scenedesmus

Summary In our earlier work we have shown that hydrogen photoproduction by photosystem I of Scenedesmus does not require O₂ evolution or cyclic photophosphorylation but must be due to non-cyclic electron flow from organic substrate(s) through photosystem I to hydrogenase, where molecular H₂ is released. The kinetics of this reaction are rather complex, in that H₂ photoproduction by Scenedesmus evidently occurs in two phases: a rapid initial phase which depends upon the dehydrogenation of a pool of H donors, and a later and slower second phase which is limited by the flow of electrons from fermentation. When adapted cells were incubated in the dark with an inhibitor (Cl-CCP or salicylaldoxime), the pool utilized by photosystem I gradually disappeared. However, the pool gave a rapid rate of hydrogen photoproduction when the adapted cells were illuminated immediately after adding the inhibitor. The rate at which the pool was utilized depended upon the light intensity and was not light-saturated at the highest intensity tested (3.4×10³ W cm^-2).With light of at least medium intensity (1.67×10³ W cm^-2), the pool was rapidly exhausted and the reaction became dependent upon the leak of electrons from fermentation. The size of the leak was found to depend upon the level of reduced organic compounds in the cell, since this process was depressed by starving the cells and was much enhanced by adding glucose or by growing the cells heterotrophically. A quantitative relationship was found between the amount of glucose added and the resulting stimulation of H₂ photoproduction, in that one mole of glucose gave about 0.5 mole of H₂ gas.The following abbreviations were used: Cl-CCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP=dichlorophenol-indophenol; PS=photosystem.These studies were supported by contract No. AT-(40-1)-2687 from the U. S. Atomic Energy Commission.     

Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

Nitrogen fixation and photoproduction of molecular hydrogen by Thiorhodaceae

Summary Nitrogen fixation has been shown to be a characteristic of two previously untested strains of the purple sulfur bacteriumChromatium sp.Chromatium strains have been shown to produce molecular hydrogen when suppliedD-L malate and bicarbonate in the presence of light and the absence of exogenous ammonia and molecular nitrogen. These results are discussed in relation to current findings on the nitrogen metabolism of the photosynthetic bacteria. Supported in part by grants from the Rockefeller Foundation, the Atomic Energy Commission, and the Research Committee of the Graduate School from funds provided by the Wisconsin Alumni Research Foundation.     

Effect of convection in capillaries on oxygen removal from arterioles in striated muscle

Based on experimental data that show the presence of significant oxygen saturation gradients in precapillary arterioles, it has been suggested that the in vivo permeability to oxygen of resting striated muscle may be significantly higher than the corresponding in vitro value obtained in unperfused tissue samples (Popel et al., 1989b, Adv. expl. Med. Biol. 247, 215). The present study performs two analyses to further compare theoretical predictions with experimental data obtained under control conditions and during hemodilution and hemoconcentration. First, it is shown that, in principle, a capillary-perfused tissue layer with a thickness of a few hundred microns is necessary to convectively carry the experimentally determined amount of oxygen released by precapillary arterioles under control and hemodiluted conditions. This capacity to convect oxygen depends strongly on the resting tissue oxygen tension. Second, a more general version of a previous model (Weerappuli & Popel, 1989, J. Biomech. Eng. 111, 24) is used to examine whether changes made in the model parameters within the physiological range of values can explain the experimentally measured flux. The results show that the theoretical predictions can be made compatible with experimental observations if the in vivo permeability of perfused tissue to oxygen is assumed to be one to two orders of magnitude higher than the in vitro value. Furthermore, the predicted in vivo permeability for perfused tissue surrounding an arteriole varies with the arteriolar luminal oxygen tension and flow. This may be due to simplifying approximations made in the model or possible experimental artifacts. Alternatively, it could also be speculated that this variability indicates the flow dependency of the permeability of perfused tissue to oxygen.     

Effect of light intensity and thickness of culture solution on oxygen production by algae

Data from a small cylindrical culture unit with variable annular culture chambers indicate that (i) the rate of oxygen evolution by an algal culture in the linear phase of growth is a logarithmic function of light intensity, and (ii) the rate of oxygen evolution per unit volume of suspension is linearly related to the reciprocal of culture thickness. These two relationships have been combined in an empirical equation which gives the expected variation of the oxygen production rate with light intensity, culture thickness, and suspension volume. The applicability of this equation has been tested on a larger, multilight culture unit in this laboratory. The agreement between the experimental and calculated oxygen production rates was very satisfactory, suggesting that the equation is not limited to a particular culture unit but may have wide applicability. The efficiency of the culture unit from the standpoint of oxygen output (chemical energy) relative to electrical energy to supply the light source has been calculated, and the maximum value of 0.51% was obtained. The energy to run auxiliary equipment was not a factor in these calculations. The maximum efficiency in converting light energy to chemical energy was approximately 12%. An extrapolation of the experimental results suggests that approximately 2 ft³ and 30 kw would be required to provide the oxygen needs of one man.     

Effect of culture medium on hydrogen production by sulfur-deprived marine green algae Platymonas subcordiformis

To study the effect of culture medium on hydrogen production by the marine green algae, Platymonas subcordiformis under sulfur deprivation, cell growth, hydrogen production, and starch and protein catabolism was investigated in the work. Algae cells cultured only in optimized medium required 6～8 days to reach the late logarithmic at the approximate density of (2.00 ± 0.18) × 10⁶ cells/mL, which in traditional medium needed 18～22 days to reach (1.85 ± 0.20) × 10⁶ cells/mL. Increased levels of Chlorophyll (10.74 ± 0.20 μg/mL), starch (149.50 ± 6.15 μg/mL), and protein (213.00 ± 7.36 μg/mL) were accumulated in optimized medium, which were 1.06, 1.47, and 1.87-fold of the algae cells cultured in traditional medium, respectively. The sealed culture of algae cells in sulfur-deprived optimized medium shifted to anaerobic conditions after 96 h of light illumination and produced 0.45 ± 0.12 mL H₂, but in traditional medium maintained aerobic condition and no hydrogen was produced. In addition, changes in starch and protein content during continuous light illumination indicated that more endogenous substrate was consumed in the sulfur-deprived optimized medium than that in the sulfur-deprived traditional medium.     

Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction

A photobioreactor was constructed with porous glass as an immobilization matrix. The reactor was a rectangular glass chamber (inner dimensions: 125 × 50 × 2.5 mm) containing a porous glass sheet (125 × 50 × 0.5 mm) on which Rhodobacter sphaeroides RV was immobilized (11.2 mg dry wt./ml porous glass). The maximum rate of hydrogen evolution was 1.3 ml/h/ml porous glass. The conversion efficiency of succinate into hydrogen reached 75%. Stable and efficient hydrogen evolution continued for up to 40 d.     

Effect of oxygen and temperature on the efficiency of photosynthetic carbon assimilation in two microscopic algae

The CO₂ compensation points of Coccochloris peniocystis, a blue-green alga and Chlamydomonas reinhardtii, a green alga, were determined at pH 8.0 in a closed system by a gas chromatographic technique. The compensation point of Chlamydomonas increased markedly with temperature, rising from 0.79 microliter per liter CO₂ at 15 C to 2.5 microliters per liter CO₂ at 35 C. In contrast, the compensation point of Coccochloris at 20 C was 0.71 microliter per liter CO₂ and rose to only 0.95 microliter per liter CO₂ at 40 C.     

Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata.

Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.     

Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters

The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions.     

Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.