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     

Increase of Nitrogenase Activity in the Blue-Green Alga Nostoc muscorum (Cyanobacterium)

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under hydrogen or argon (nongrowing conditions, neither CO₂ nor N₂ or bound nitrogen present) in the light resulted in a two- to fourfold increase of light-induced hydrogen evolution and a 30% increase of acetylene reduction. Preincubation under the same gases in the dark led to a decrease of both activities. Cultivation of algae under a hydrogen-containing atmosphere (N₂, H₂, CO₂) increased neither hydrogen nor ethylene evolution by the cells. Formation of both ethylene and hydrogen is due to nitrogenase activity, which apparently was induced by the absence of N₂ or bound nitrogen and not by the presence of hydrogen. Inhibitors of protein biosynthesis prevented the increase of nitrogenase activity. Hydrogen uptake by the cells was almost unaffected under all of these conditions. With either ammonia or chloramphenicol present, nitrogenase activity decreased under growing conditions (i.e., an atmosphere of N₂ and CO₂). The kinetics of decrease were the same with ammonia or chloramphenicol, which was interpreted as being due to rapid protein breakdown with a half-life of approximately 4 h. The decay of nitrogenase activity caused by chloramphenicol could be counteracted by nitrogenase-inducing conditions, i.e., by the absence of N₂ or bound nitrogen. A cell-free system from preconditioned algae with an adenosine 5′-triphosphate-generating system exhibited the same increase or decrease of nitrogenase activity as the intact cell filaments, indicating that this effect resided in the nitrogenase complex only. We tentatively assume that not the whole nitrogenase complex, but merely a subunit or a special protein with regulatory function, is susceptible to fast turnover.     

Kinetics of sulfate uptake by freshwater and marine species ofDesulfovibrio

Apparent half-saturation constants (K _m) and maximum uptake rates (V _max) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a³⁵S-sulfate tracer technique. The lowerstK _m (5 M) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestK _m (77 M) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,V _max) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsK _m andV _max between 1 and 6,000 M SO ₄ ^2- , and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.Abbreviations MPB methane producing bacteria - SRB sulfate reducing bacteria     

Cyanide assimilation in Rhizobium ORS 571: influence of the nitrogenase catalyzed hydrogen production on the efficiency of growth

When cyanide is gradually added to a nitrogenfixing culture, Rhizobium ORS 571 is capable of assimilating large amounts of cyanide using its nitrogenase. Under these conditions the molar growth yield on succinate (Y _succ) increases from 27 at the start of cyanide addition to 38 at the end. The respiratory chain of cells grown at a concentration of 7 mM cyanide is still very sensitive to cyanide. The increase in growth yield is explained by a decrease in hydrogen production by nitrogenase as soon as cyanide is assimilated. This is confirmed by calculating the influence of hydrogen production on Y _succ. Hydrogen production by nitrogenase has a greater influence on growth yields than the presence or absence of hydrogenase activity. At the end of cyanide addition when all cell nitrogen is synthesized from cyanide and no nitrogen fixation occurs, nitrogenase will be in a very oxidized state.     

Pathways of hydrogen uptake in the cyanobacterium Nostoc muscorum

Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl chlorophyll - DCMU (diuron) 3-3,4-dichlorophenyl)-1,1-dimethylurea - pev packed cell volume     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.     

Nitrogenase and photosynthetic activities of chemostat cultures of Rhodobacter capsulatus 37b4 grown under different illuminations

Nitrogen fixation as well as structural and functional properties of the photosynthetic apparatus were studied with phototrophically grown chemostat cultures of Rhodobacter capsulatus strain 37b4. Illumination was varied between 3,000 and 30,000 lx at a constant dilution rate of D=0.075 h^-1. Steady state parameters of growth revealed two forms of limitation, i.e. energy limitation in the range of 3,000 to about 10,000 lx and nitrogen limitation at higher illuminations. Over the entire range of illumination, the specific bacteriochlorophyll content and the amount of total bacteriochlorophyll per photochemical reaction center remained essentially constant. Photophosphorylation activity remained constant up to 20,000 lx but was slightly increased at 30,000 lx. Hydrogen evolution and acetylene reduction activities of cellular nitrogenase were assayed under saturating light conditions with samples taken from cultures growing under steady state conditions. In spite of the apparent constancy of the composition and activity of the photosynthetic apparatus under energy limitation, maximal specific acetylene reduction and hydrogen evolution activities increased by factors of 3 and 8, respectively, when illumination of the culture was raised from 3,000 to about 15,000 lx. Above 15,000 lx, both activities of nitrogenase approached constancy.We, therefore, conclude that neither under energy limitation nor under nitrogen limitation the function of nitrogenase depended on the photosynthetic activities. Moreover, it is suggested that light did not influence nitrogenase activity under conditions of nitrogen limitation, while under conditions of energy limitation light seemed to influence nitrogenase activities indirectly via glutamate consumption of the cells.     

Patterns of sulfur isotope fractionation during microbial sulfate reduction

Studies of microbial sulfate reduction have suggested that the magnitude of sulfur isotope fractionation varies with sulfate concentration. Small apparent sulfur isotope fractionations preserved in Archean rocks have been interpreted as suggesting Archean sulfate concentrations of <200 μm , while larger fractionations thereafter have been interpreted to require higher concentrations. In this work, we demonstrate that fractionation imposed by sulfate reduction can be a function of concentration over a millimolar range, but that nature of this relationship depends on the organism studied. Two sulfate‐reducing bacteria grown in continuous culture with sulfate concentrations ranging from 0.1 to 6 mm showed markedly different relationships between sulfate concentration and isotope fractionation. Desulfovibrio vulgaris str. Hildenborough showed a large and relatively constant isotope fractionation (³⁴ε_SO4‐H2S ? 25‰), while fractionation by Desulfovibrio alaskensis G20 strongly correlated with sulfate concentration over the same range. Both data sets can be modeled as Michaelis–Menten (MM)‐type relationships but with very different MM constants, suggesting that the fractionations imposed by these organisms are highly dependent on strain‐specific factors. These data reveal complexity in the sulfate concentration–fractionation relationship. Fractionation during MSR relates to sulfate concentration but also to strain‐specific physiological parameters such as the affinity for sulfate and electron donors. Previous studies have suggested that the sulfate concentration–fractionation relationship is best described with a MM fit. We present a simple model in which the MM fit with sulfate concentration and hyperbolic fit with growth rate emerge from simple physiological assumptions. As both environmental and biological factors influence the fractionation recorded in geological samples, understanding their relationship is critical to interpreting the sulfur isotope record. As the uptake machinery for both sulfate and electrons has been subject to selective pressure over Earth history, its evolution may complicate efforts to uniquely reconstruct ambient sulfate concentrations from a single sulfur isotopic composition.     

Temporal separation of nitrogen fixation and photosynthesis in the filamentous,non-heterocystous cyanobacterium Oscillatoria sp.

When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C₂H₄ and 1.4 mg O₂ mg Chl a ^-1·h^-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C₂H₄ mg Chl a ^-1·h^-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.     

Hydrogen uptake by Robinia pseudoacacia nodules

Summary Hydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H₂ produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae.     

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.     

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     

Relative efficiency of nitrogen fixation and the occurrence of hydrogenase in pea root nodules

Summary The apparent K_m(hydrogen) for uptake of hydrogen by pea root nodules was determined. This enabled the concentration gradient necessary for the evolution of hydrogen to be calculated for nodules with no hydrogenase activity. This indicated that hydrogen inhibition of nitrogenase is not likely to be the cause of the low relative efficiency of legume root nodules. The factors that affect electron allocation between protons and nitrogen in nitrogenase are reviewed and it is concluded that there must be some as yet unknown factor that affects electron distribution inRhizobium nitrogenase. One possibility is put forward and considered. A strain ofRhizobium was used that was found to possess hydrogenase activity in combination with pea variety Feltham First but not with variety Meteor. The control of this enzyme is briefly discussed.     

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.     

Isolation and characterization of Desulfovibrio senezii sp. nov., a halotolerant sulfate reducer from a solar saltern and phylogenetic confirmation of Desulfovibrio fructosovorans as a new species

A new halotolerant Desulfovibrio, strain CVL^T (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVL^T had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H₂/CO₂ + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVL^T is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436). Received: 27 February 1998 / Accepted: 15 June 1998     

Aerobic Hydrogen Accumulation by a Nitrogen-Fixing Cyanobacterium, Anabaena sp

Hydrogen evolution by a nitrogen-fixing cyanobacterium, Anabaena sp. strain N-7363, was tested in order to develop a water biophotolysis system under aerobic conditions. A culture of the strain supplemented with carbon dioxide under an air atmosphere evolved hydrogen and oxygen gas, which reached final concentrations of 9.7 and 69.8%, respectively, after 12 days of incubation. Hydrogen uptake activity was not observed during incubation, and nitrogenase was thought to be the sole enzyme responsible for the hydrogen evolution.     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.