Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

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.     

Environmental factors affectingin vitro nitrogenase activity of cyanobacteria isolated from rice-fields

The nitrogen-fixing capacity of four cyanobacterial strains was tested in relation to heterotrophic ability, tolerance to combined nitrogen and adaptive capacity to changes in light intensity and pH. Strains (Anabaena variabilis UAM 202;Calothrix marchica UAM 214;Nodularia spumigena UAM 204,Nostoc punctiforme UAM 205) were isolated from the rice-fields of Valencia (Spain).C. marchica, was the only strain able to grow and to fix dinitrogen under heterotrophic conditions, with fructose and glucose. Fructose was the best substrate supporting growth and dinitrogen fixation in mixotrophy (growth in the light under conditions where CO₂ and organic carbon are assimilated simultaneously), photoheterotrophy (growth in the light on an organic compound in the absence of net CO₂ fixation) and heterotrophy (growth on an organic compound in the dark). Ammonium repressed nitrogenase more than nitrate. Full repression was observed only at concentrations of combined nitrogen higher than those usually found in rice-fields. Carbohydrates had a protective effect on nitrogenase against ammonium inhibition inC. marchica. All four strains showed increased nitrogenase activity when the light intensity was increased during assays. Variations of pH normally occurring in rice fields led to no important changes in nitrogenase activity inC. marchica. This fact, together with its potential for heterotrophic growth and tolerance to combined nitrogen, make this the most suitable of the four strains for inoculation experiments in rice fields.     

Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

The filamentous, non-heterocytous, nitrogen-fixing cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions nitrogenase activity is exclusively present during the dark period. However, if anaerobic conditions were applied during the dark period, two activity maxima were observed. A relatively small activity peak occurred during the first few hours of the dark period and a high peak as soon as the light was switched on. A low activity remained during the second half of the dark period. This pattern of acetylene reduction in Oscillatoria agrees well with the field data on nitrogen fixation [Stal, L.J. and Krumbein, W.E. (1984), Mar. Biol. 82, 217–224].     

Identification of a nuclease and host restriction-modification in the unicellular, aerobic nitrogen-fixing cyanobacterium Cyanothece sp.

In the process of developing a gene transfer system for the marine, unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A cell wall-associated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cyanothece sp. strain BH68K chromosomal DNA. The nuclease is easily released from intact cells by using water or buffer containing Triton X-100. Nuclease activity was undetectable in cell extracts prepared from water-washed cells. Comparison of the restriction endonuclease susceptibility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. strain PCC 7120 revealed that these organisms have a nearly identical pattern of restriction and therefore may contain similar systems for DNA methylation. Restriction by DpnI, MboI, and Sau3AI indicated the presence of adenine methylation. Cyanothece sp. strain BH68K cell extracts contain a type II restriction endonuclease, Csp68KI. The activity of Csp68KI was easily detected in cell extracts without extensive purification. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucleotides producing 3-bp 5' overhang ends.     

GROWTH,PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM,CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N₂ fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O₂-sensitive N₂ fixation from photosynthetic O₂ evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.     

Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria

Abstract The enzyme activities responsible for the evolution and consumption of hydrogen in three unicellular cyanobacteria were investigated. Gloeothece sp. 6909 and Cyanothece sp. 7822 performed an oxygen-tolerant nitrogen fixation, whereas the nitrogenase activity of Synechococcus sp. 7425 was much more sensitive to oxygen. While in Gloeothece the net hydrogen production during nitrogen fixation was relatively low due to recycling by an uptake hydrogenase, little hydrogen consumption was detected in Cyanothece and Synechococcu . On the other hand a reversible hydrogenase was demonstrated in the latter strains. However, only Cyanothece shows hydrogenase-catalysed hydrogen production in vivo under anaerobic conditions in the dark. It is suggested that hydrogen is a fermentation product, and that the physiological function of this reversible hydrogenase is the removal of excess reduction equivalents under such conditions.     

Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142.

It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.     

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Acetylene reduction (nitrogen fixation) by enterobacteriaceae isolated from paper mill process waters

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.     

Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation.

A recombinant plasmid encoding Rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid pRK290:4:46) (E. Bolton, B. Higgisson, A. Harrington, and F. O'Gara, Arch. Microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum. The expression of the dct sequences on plasmid pRK290:4:46 in B. japonicum CJ1 resulted in increased growth rates in media containing dicarboxylic acids as the sole source of carbon. In addition, strain CJ1(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions. Under free-living nitrogen-fixing conditions, strain CJ1(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity compared with that of the wild-type strain. This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions. The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains. The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed.     

Nitrogen fixation and ammonia switch-off in the photosynthetic bacterium Rhodopseudomonas viridis.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Oxygen sensitivity of nitrogenase is not always a limiting factor of growth under nitrogen-fixing conditions inAzotobacter vinelandii

Summary The effect of a combined nitrogen source to oxygen sensitive mutants was examined. For some oxygen sensitive mutants, oxygen sensitivity was not restored by the addition of nitrogen compounds to their medium. One of these mutants showed oxygen resistant nitrogenase activity similar to that of a wild strain. Results imply that oxygen sensitivity of nitrogenase is not always a limiting factor of growth under aerobic nitrogen-fixing conditions inAzotobacter vinelandii.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.