Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na⁺ on the metabolism of cyanobacteria with emphasis on the N₂,fixing filamentous species. Na⁺ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na⁺ is specific and cannot be replaced by K⁺, Li⁺, Ca 2 + or Mg²⁺. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na⁺. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na⁺ but the enzyme complex is catalytically inactive. Photoevolution of O₂ and CO₂ fixation, which are severely inhibited in the absence of Na⁺, are quickly restored by glutamine or glutamate indicating that Na⁺ deprivation affects photosynthesis indirectly due to deficiency in the products of N₂ fixation. Na⁺ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na⁺ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na⁺ starvation. Na⁺ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K⁺ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na⁺ influx respectively. These cyanobacteria exhibit rapid active efflux of Na⁺, in a manner different from the Na⁺/H⁺ antiporter mechanism found inAnacystis nidulans. Na⁺ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.     

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D₂ uptake and in H₂ and HD evolution. In contrast to the nitrogenase-mediated H₂ production, this anaerobic H₂ and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H₂ and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D₂ is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.     

Nitrogenase activity of the antarctic cyanobacteriumNostoc commune: Influence of temperature

Nitrogenase activity (C₂H₂ reduction) ofNostoc commune isolated from Schirmacher Oasis (Antarctica) was compared withNostoc muscorum, N. calcicola, Anabaena doliolum andGloeocapsa sp. The temperature profile of acetylene reduction (5–30 °C) forN. commune revealed (a) that the highest rate of nitrogenase activity was at 25±1 °C, (b) that it was low (69 %) in comparison withN. muscorum, and (c) that nitrogenase activity continued at lower temperatures, which was not evident for other cyanobacteria. The results suggest thatN. commune is adapted to lower temperatures in terms of nitrogen fixation.     

Acetylene reduction and hydrogen metabolism by a cyanobacterial/sulfate‐reducing bacterial mat ecosystem

We report a study of nitrogenase activity (acetylene reduction) and hydrogen gas metabolism in intact smooth cyanobacterial mats from Hamelin Pool, Shark Bay, Western Australia. The predominant cyanobacterial population in these mats is Microcoleus chthonoplastes. The mats had a significant capacity for nitrogen fixation, predominantly attributable to the photosyn‐thetic component. By physical and chemical perturbation we revealed an active hydrogen metabolism within the mats. Most of the H₂ formation was attributed to fermentative processes, whereas hydrogen was consumed in light‐dependent, together with oxygen‐ and sulfate‐dependent respiratory processes. It was concluded that H₂ formed by fermentative bacteria in the dark drives a significant proportion of sulfate reduction in the mats, but there was little H₂ transfer from the cyanobacteria to the sulfate‐reducing bacteria. Thus photosynthetically produced H₂ gas is unlikely to significantly alter the previously measured carbon: sulfur ratio relating photosynthesis to sulfate reduction.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

Diversity of Cyanobacterial Hydrogenases, a Molecular Approach

In an effort to elucidate the diversity of cyanobacterial hydrogenases, we used a molecular approach. Filamentous strains from a broad range of sources were screened for the presence of hup (uptake hydrogenase), xisC (rearrangement within hupL), and hox (bidirectional hydrogenase) genes. As expected, an uptake hydrogenase seems to be present in all N₂-fixing cyanobacteria. On the other hand, no evidence was found for the presence of a conventional bidirectional enzyme in several strains. Similarly, the presence of xisC is not a characteristic shared by all the heterocyst-forming cyanobacteria. Although tempting, it is not possible to establish a correlation between the presence/absence of the bidirectional hydrogenase and the occurrence of xisC. The natural molecular variation of hydrogenases in cyanobacteria is certainly a field to explore, both to understand the physiological functions of the respective enzymes and to identify a genetic background to be used when constructing a strain for photobiological H₂ production in a bioreactor. Received: 3 November 1999 / Accepted: 8 December 1999     

Interspecific protection against oxidative stress: green algae protect harmful cyanobacteria against hydrogen peroxide

Oceanographic studies have shown that heterotrophic bacteria can protect marine cyanobacteria against oxidative stress caused by hydrogen peroxide (H₂O₂). Could a similar interspecific protection play a role in freshwater ecosystems? In a series of laboratory experiments and two lake treatments, we demonstrate that freshwater cyanobacteria are sensitive to H₂O₂ but can be protected by less-sensitive species such as green algae. Our laboratory results show that green algae degrade H₂O₂ much faster than cyanobacteria. Consequently, the cyanobacterium Microcystis was able to survive at higher H₂O₂ concentrations in mixtures with the green alga Chlorella than in monoculture. Interestingly, even the lysate of destructed Chlorella was capable to protect Microcystis, indicating a two-component H₂O₂ degradation system in which Chlorella provided antioxidant enzymes and Microcystis the reductants. The level of interspecific protection provided to Microcystis depended on the density of Chlorella. These findings have implications for the mitigation of toxic cyanobacterial blooms, which threaten the water quality of many eutrophic lakes and reservoirs worldwide. In several lakes, H₂O₂ has been successfully applied to suppress cyanobacterial blooms. Our results demonstrate that high densities of green algae can interfere with these lake treatments, as they may rapidly degrade the added H₂O₂ and thereby protect the bloom-forming cyanobacteria.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Biohydrogen production from carbon monoxide and water byRhodopseudomonas palustris P4

A reactor-scale hydrogen (H₂) productionvia the water-gas shift reaction of carbon monoxide (CO) and water was studied using the purple nonsulfur bacterium,Rhodopseudomonas palustris P4. The experiment was conducted in a two-step process: an aerobic/chemoheterotrophic cell growth step and a subsequent anaerobic H₂ production step. Important parameters investigated included the agitation speed, inlet CO concentration and gas retention time. P4 showed a stable H₂ production capability with a maximum activity of 41 mmol H₂ g cell⁻¹h⁻¹ during the continuous reactor operation of 400 h. The maximal volumetric H₂ production rate was estimated to be 41 mmol H₂ L¹h⁻¹, which was about nine-fold and fifteen-fold higher than the rates reported for the photosynthetic bacteriaRhodospirillum rubrum andRubrivivax gelatinosus, respectively. This is mainly attributed to the ability of P4 to grow to a high cell density with a high specific H₂ production activity. This study indicates that P4 has an outstanding potential for a continuous H₂ productionvia the water-gas shift reaction once a proper bioreactor system that provides a high rate of gas-liquid mass transfer is developed.     

Energization and activation of inorganic carbon uptake by light in cyanobacteria

The requirement of the inorganic carbon (C_i) transport system for light in cyanobacteria was investigated in Anabaena variabilis by the filtering centrifugation technique and in a mutant (E₁) isolated from Anacystis nidulans using a gas exchange system. C_i transport capability increased with time of preillumination and decreased following darkening. Full activity could not be obtained by operating either photosystem II (PSII) or photosystem I alone. 3(3,4 Dichlorophenyl)-1,1 dimethylurea strongly inhibited C_i uptake. Very low activity of PSII was sufficient to activate C_i uptake. However, in the presence of dithiothreitol PSII activity was not required. We conclude that light may be required to activate as well as to energize C_i uptake in cyanobacteria.     

Effects of growth conditions of acetate utilization byRhodopseudomonas palustris isolated from a freshwater lake

Rhodopseudomonas palustris, a purple non-sulfur bacterium, was recently found throughout the water column in Lake Kinneret. It was demonstrated to be of a versatile nature, growing under both aerobic and anaerobic conditions at different light intensities. A comparison of C-acetate uptake byR. palustris andChlorobium phaeobacterioides, a green sulfur bacterium, showed that, under identical growth conditions, C-acetate assimilation byR. palustris was greater. Furthermore, C-acetate uptake forR. palustris was greater than C−CO₂ uptake at all light intensities. Depending on the prevailing conditions, acetate can be used byR. palustris as both an electron donor and carbon source. Malate synthase was used as an indicator of activity of the glyoxylic acid cycle. It was found that enzyme activity was higher (i.e., acetate was used mainly as a carbon source) under anaerobic conditions, in the dark, or in the absence of HCO ₃ ⁻ . Acetate was used preferably as an electron donor under photosynthetic microaerophillic conditions.     

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.     

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H₂ utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H₂ uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H₂ uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H₂ metabolism.     

Relationship of soil hydrogen sulfide level to net carbon assimilation ofPanicum hemitomon andSpartina patens

Panicum hemitomon Schult andSpartina patens (Ait) Muhl. plants from Louisiana Gulf Coast fresh and brackish marshes were subjected to hydrogen sulfide under controlled sediment redox conditions. Net carbon assimilation responses of both species to the combined sediment anaerobiosis and hydrogen sulfide concentrations was measured.Panicum hemitomon was more sensitive to hydrogen sulfide as compared toSpartina patens. Initiation of reduction in net carbon assimilation inP. hemitomon began when H₂S concentrations of soil solution exceeded 0.22 mgl^-1. Reductions in net carbon assimilation inS. patens were also noted at H₂S concentrations exceeding 0.34 mgl^-1. The reduction in net carbon assimilation of both species measured at elevated H₂S concentrations suggests that extreme anaerobiosis and elevated sulfide could contribute to the growth reduction of these species under certain conditions. However based on H₂S concentration in fresh and brackish marsh soil profiles, levels were too low to cause any adverse effects ofPanicum hemitomon. In brackish marsh soils containing hydrogen sulfide of 3.4 mgl^-1 in soil solution, sulfide could be a major factor limiting growth ofS. patens.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

A simplified method for assay of hydrogenase activities of H<Subscript>2</Subscript> evolution and uptake in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis>

Assay of hydrogenase activity pertaining to H₂ production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H₂ uptake and evolution activities of the intact cells, respectively. This method enabled H₂ uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H₂ uptake hydrogenase activity in E. aerogenes.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 12 April 2005 and 17 May 2005