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.     

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.     

The cell content and secretion of water-soluble vitamins by several freshwater algae

Three green algae, Chlamydomonas reinhardii, Chlorella vulgaris and Scenedesmus obliquus, and one blue-green alga, Anabaena cyclindrica, were grown in chemically defined media. All the algac examined contained folates, -carotene and vitamins C and E; several of the B-vitamins and vitamin A were found in varying amounts in some but not in all the algae examined. All the green algae secreted significant amounts of folate and biotin and all but Scenedesmus secreted pantothenate into their growth medium; Anabaena secreted folate and pantothenate.This work was done with the support of grant BMS 74-08918 from the National Science Foundation     

绿藻光合生物制氢技术进展

氢能作为可再生、环境友好的能源,已成为营造可持续发展的经济节约型社会的理想能源。绿藻因能利用光能分解水产氢,被称为最有应用前景的方法之一。本文将综述绿藻光合产氢的原理,介绍该生物制氢技术的研究现状和最新进展,并对其发展趋势做以展望。     

Ascorbate as a substrate for photoproduction of hydrogen by photosystem I of chloroplasts

The photoproduction of hydrogen by 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts from ascorbate under anaerobic conditions was studied in the pH range 5.0 to 7.5 using methyl viologen (MV), N,N,N′,N′-tetramethyl-P-phenylenediamine (TMPD), and excess hydrogenase from Desulfovibrio desulfuricans. (a) At neutral and basic pHs, the photoreduction of MV, which reacted back with photoxidized ascorbate (dehydroascorbate [DHASC]), and the rates of H₂ photoproduction were very low. The slow H₂ photoproduction was explained by the reversible reduction of MV by the photoproduced H₂ (catalyzed by hydrogenase) and its reoxidation by DHASC resulting in H₂ uptake. (b) At pH 5.2, relatively high initial rates of H₂ photoproduction were obtained, which were comparable to the rates of O₂ consumption at pH 5.2 by photosystem I (catalyzed by photoreduced MV). However, accumulation of photoreduced MV under anaerobic conditions was not detected. In the presence of high concentrations of protons, the H₂ uptake by DHASC was very slow because the equilibrium concentration of H₂-reduced MV was very small, thus allowing H₂ evolution mediated by photoreduced MV to compete with the back reaction with DHASC. (c) The continuous accumulation of DHASC, which was generated together with H₂, gradually slowed the H₂ evolution until it stopped after about 3 hours. At high concentrations, DHASC was able to compete with the coupling of photoreduced MV to hydrogenase and H₂ evolution. (d) Dithiothreitol (DTT) reduced the DHASC and consequently competed with the back reaction of the photoreduced and H₂-reduced MV with DHASC. DTT thus prolonged the time period of H₂ photoproduction from ascorbate and abolished the dependence of its rate on pH in the range of 5.2 to 7.5 (e) A study of H₂ uptake by chemically oxidized ascorbate (in the dark) showed that MV and hydrogenase were both required to catalyze electron transfer from H₂ to DHASC. TMPD prevented this H₂ consumption by DHASC (in a chloroplast reaction mixture containing MV and hydrogenase). Illumination restored the H₂ uptake presumably by generating reduced MV which activated the hydrogenase.     

A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions

Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production.     

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.     

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.     

Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus

The influence of (NH₄)₂SO₄ concentration and dilution rate (D) on actual and potential H₂ photoproduction has been studied in ammonium-limited chemostat cultures of Rhodobacter capsulatus B10. The actual H₂ production in a photobioreactor was maximal (approx. 80 ml h⁻¹l⁻¹) at D = 0.06 h⁻¹ and 4 mM (NH₄)₂SO₄. However, it was lower than the potential H₂ evolution (calculated from hydrogen evolution rates in incubation vials), which amounted to 100–120 ml h⁻¹ l⁻¹ at D = 0.03–0.08 h⁻¹. Taking into account the fact that H₂ production in the photobioreactor under these conditions was not limited by light or lactate, another limiting (inhibiting) factor should be sought. One possibility is an inhibition of H₂ production by the H₂ accumulated in the gas phase. This is apparent from the non-linear kinetics of H₂ evolution in the vials or from its inhibition by the addition of H₂; initial rates were restored in both cases after the vials had been refilled with argon. The actual H₂ production in the photobioreactor at D = 0.06 h⁻¹ was shown to increase from approximately 80 ml h⁻¹ l⁻¹ to approximately 100 ml h⁻¹ l⁻¹ under an argon flow at 100 ml min⁻¹. Under maximal H₂ production rates in the photobioreactor, up to 30% of the lactate feedstock was utilised for H₂ production and 50% for biomass synthesis. Received: 22 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

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.     

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.     

pEffects of UV radiation on the ultrastructure of several red algae

The effect of ultraviolet (UV) radiation on the ultrastructure of four red algae, the endemic Antarctic Palmaria decipiens (Reinsch) Ricker and Phycodrys austrogeorgica Skottsberg, the Arctic‐cold temperate Palmaria palmata (Linnaeus) O. Kuntze and the cosmopolitan Bangia atropurpurea (Roth) C. Agardh was studied. All four species showed a formation of ‘inside‐out’ vesicles from the chloroplast thylakoids upon exposure to artificial UV‐radiation. In P. decipiens, most vesicles were developed after 8 h and in P. palmata, after 48 h of UV exposure. In B. atropurpurea, vesi‐culation of thylakoids was observed after 72 h of UV irradiation. In Ph. austrogeorgica, the chloroplast envelope and thylakoid membranes were damaged and the phycobilisomes became detached from the thylakoids after 12 h of UV exposure. Ultraviolet‐induced changes in the membrane structure of mitochondria were observed in P. decipiens and P. palmata. However, in P. decipiens they were reversible as was the damage in chloroplast fine structure after 12 h of UV treatment. Protein crystals in Ph. austrogeorgica showed degradation after exposure to UV radiation. Different methods of fixation and embedding macroalgal material are discussed. These findings give insight into the fine structural changes which occur during and after UV exposure and indicate a relationship between the species dependent sensitivity to UV‐exposure and the depth distribution of the different species.     

Observations on interactions between naturally-collected bacteria and several species of algae

Interactions between a naturally-collected algal species and strains of bacteria with which it was closely associated were examined under controlled conditions. Three strains of bacteria, Pseudomonas, Xanthomonas and Flavobacterium, were isolated from Oscillatoria. These bacteria were grown in combination with axenic cultures of the Oscillatoria culture as well as with several additional algal species. Oscillatoria growth was stimulated by all of the bacteria, but other algal species varied in their response. Some were stimulated, but others were inhibited or unaffected by exposure to the bacterial strains. There were also observations indicating that some algae may be able to develop resistance to antagonistic bacteria. These data suggest that succession and dominance of individual algal species may be influenced by interactions with bacteria.     

Effects of temperature on the production of hydrogen peroxide and volatile halocarbons by brackish-water algae

Marine algae produce volatile halocarbons, which have an ozone-depleting potential. The formation of these compounds is thought to be related to oxidative stress, involving H2O2 and algal peroxidases. In our study we found strong correlations between the releases of H2O2 and brominated and some iodinated compounds to the seawater medium, but no such correlation was found for CHCl3, suggesting the involvement of other formation mechanisms as well. Little is known about the effects of environmental factors on the production of volatile halocarbons by algae and in the present study we focused on the influence of temperature. Algae were sampled in an area of the brackish Baltic Sea that receives thermal discharge, allowing us to collect specimens of the same species that were adapted to different field temperature regimes. We exposed six algal species (the diatom Pleurosira laevis, the brown alga Fucus vesiculosus and four filamentous green algae, Cladophora glomerata, Enteromorpha ahlneriana, E. flexuosa and E. intestinalis) to temperature changes of 0-11 degrees C under high irradiation to invoke oxidative stress. The production rates, as well as the quantitative composition of 16 volatile halocarbons, were strongly species-dependent and different types of responses to temperature were recorded. However, no response patterns to temperature change were found that were consistent for all species or for all halocarbons. We conclude that the production of certain halocarbons may increase with temperature in certain algal species, but that the amount and composition of the volatile halocarbons released by algal communities are probably more affected by temperature-associated species shifts. These results may have implications for climatic change scenarios.