Effect of Boron Deficiency on Photosynthesis and Reductant Sources and Their Relationship with Nitrogenase Activity in Anabaena PCC 7119

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O₂ evolution and in CO₂ fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N₂ fixation is independent of its effects on photosynthesis and reductant supply.     

Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed

Some unicellular N₂-fixing cyanobacteria have recently been found to lack a functional photosystem II of photosynthesis. Such organisms, provisionally termed UCYN-A, of the oceanic picoplanktion are major contributors to the global marine N-input by N₂-fixation. Since their photosystem II is inactive, they can perform N₂-fixation during the day. UCYN-A organisms cannot be cultivated as yet. Their genomic analysis indicates that they lack genes coding for enzymes of the Calvin cycle, the tricarboxylic acid cycle and for the biosynthesis of several amino acids. The carbon source in the ocean that allows them to thrive in such high abundance has not been identified. Their genomic analysis implies that they metabolize organic carbon by a new mode of life. These unicellular N₂-fixing cyanobacteria of the oceanic picoplankton are evolutionarily related to spheroid bodies present in diatoms of the family Epithemiaceae, such as Rhopalodia gibba. More recently, spheroid bodies were ultimately proven to be related to cyanobacteria and to express nitrogenase. They have been reported to be completely inactive in all photosynthetic reactions despite the presence of thylakoids. Sequence data show that R. gibba and its spheroid bodies are an evolutionarily young symbiosis that might serve as a model system to unravel early events in the evolution of chloroplasts. The cell metabolism of UCYN-A and the spheroid bodies may be related to that of the acetate photoassimilating green alga Chlamydobotrys.     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.     

N2 fixation in phototrophs: adaptation to a specialized way of life

Phototrophic diazotrophs include the photosynthetic green and purple bacteria, the heliobacteria, many cyanobacteria and the unusual chlorophyll-containing rhizobia that are found in the stem nodules of Aeschynomene spp. In this review, which concentrates on cyanobacteria, the interrelations between photosynthesis and N₂ fixation are discussed. Photosynthesis can, in theory, directly provide the ATP and reductant needed to support N₂ fixation but the link between these two processes is usually indirect, mediated through accumulated carbon reserves. In cyanobacteria, which possess an oxygenic photosynthesis, this serves to separate the O₂ that is produced by photosynthesis from the O₂-sensitive nitrogenase. However, in certain circumstances, oxygenic photosynthesis and N₂ fixation coexist. Under these conditions, respiratory consumption of photosynthetically generated O₂ may have an important role in minimizing O₂-damage to nitrogenase.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique

1. The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C₃-alga Chlamydomonas reinhardii were determined by measuring the ratio of ¹³CO₂ to ¹²CO₂ incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C₃-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO₂ mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.Abbreviations RuDP ribulose 1,5-disphosphate - PEP phosphoenolpyruvate     

Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O₂ pressure (pO₂) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary “switching” of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.     

Photosynthesis in Rhodospirillum rubrum. III. Metabolic Control of Reductive Pentose Phosphate and Tricarboxylic Acid Cycle Enzymes

Enzymes of the reductive pentose phosphate cycle including ribulose-diphosphate carboxylase, ribulose-5-phosphate kinase, ribose-5-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose-1,6-diphos-phatase were shown to be present in autotrophically grown Rhodospirillum rubrum. Enzyme levels were measured in this organism grown photo- and dark heterotrophically as well. Several, but not all, of these enzymes appeared to be under metabolic control, mediated by exogenous carbon and nitrogen compounds. Light had no effect on the presence or levels of any of these enzymes in this photosynthetic bacterium.

The enzymes of the tricarboxylic acid cycle and enolase were shown to be present in R. rubrum cultured aerobically, autotrophically, or photoheterotrophically, both in cultures evolving hydrogen and under conditions where hydrogen evolution is not observed. Light had no clearly demonstrable effect on the presence or levels of any of these enzymes.

     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged     

The pathway of carbon dioxide assimilation in rhodospirillum rubrum grown in turbidostat continuous-flow culture

Summary A comparison of light and dark short-term incorporation of [¹⁴C]-carbon dioxide by Rhodospirillum rubrum grown in turbidostat continuous-flow culture at two different steady states on medium containing malate has shown that the labelling of phosphate esters was the main light-dependent process. Thus, the reductive pentose phosphate cycle appears to be the major pathway of carbon dioxide assimilation in the light under these growth conditions.The labelling of glutamate was also light-dependent and was most marked in the most rapidly growing steady state culture.The assimilated [¹⁴C]carbon was transferred to metabolites of the tricarboxylic acid cycle, particularly C₄-dicarboxylic acids, and the transfer involved additional carboxylations which were not light-dependent. The activity of these reactions accounted for initial high rates of carbon dioxide assimilation in the dark.In the dark assimilated [¹⁴C]carbon accumulated in succinate.     

Light‐dependent oxygen consumption in nitrogen‐fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O₂ through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O₂, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O₂. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer ¹⁸O₂ and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O₂ consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O₂ concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N₂‐fixing Trichodesmium colonies, Mehler activity can consume ～75% of gross O₂ production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ～10% of gross O₂ production. Moreover, evidence for the coupling between N₂ fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O₂ consumption by 3‐fold. Our results suggest that a major role for PSI in N₂‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O₂ through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.     

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N₂-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N₂-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N₂-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N₂-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.Abbreviations Chl chlorophyll - EDTA ethylenediaminetetraacetic acid - MSX l-methionine-d,l-sulfoximine     

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)^-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO₂-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO₂-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO₂ release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Electron transport to nitrogenase in Rhodospirillum rubrum: the role of NAD(P)H as electron donor and the effect of fluoroacetate on nitrogenase activity

The role of the reactions of the TCA cycle in the generation of reductant for nitrogenase in Rhodospirillum rubrum has been investigated. Addition of fluoroacetate inhibited nitrogenase activity almost completely when pyruvate or endogenous sources were used as electron donors, whereas the inhibition was incomplete when malate, succinate or fumarate were used. Addition of NAD(P)H to cells supported nitrogenase activity, both with and without prior addition of fluoroacetate. We suggest that the role of the TCA cycle in nitrogen fixation in R. rubrum is to generate reduced pyridine nucleotides which are oxidized by the components of the electron transport pathway to nitrogenase.     

Properties of heterocysts isolated with colloidal silica

A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and Na₂S₂O₄ reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13% of the activity of the intact filaments was accounted for in the isolated heterocyst preparation.Isolated heterocysts took up O₂ in a light-independent process; O₂ uptake with added NADP⁺ was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Cellular differentiation in the process of generation of the eukaryotic cell

Primitive atmosphere of the earth did not contain oxygen gas (O₂) when the proto-cells were generated successfully as the resut of chemical evolution and then evolved. Therefore, they first had acquired anaerobic energy metabolism, fermentation. The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions. Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle. On the other hand, O₂-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem. These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacteriumChlorogloea fritschii and aerobic photosynthetic bacteriaRhodospirillum rubrum andErythrobacter sp. Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium. Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O₂ -respiratory enzymes show similarities to corresponding prokaryotic enzymes. However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules. In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only in phylogenetically close relationships but also under highly selective pressure, that is under a physiological constraint for the species in their habitats. Therefore, the similarities in amino acid sequences of proteins between the prokaryotes and the organelles are not necessarily direct evidence for their phylogenetical closeness: it gives still less evidence for a symbiotic relationship between the prokaryotes and the organelles. The metabolic compartmentalization of the membranes is an important tendency in cellular evolution to guarantee high specificity and rate of the metabolisms. It is suggested from the data that the intracellular membranes are not static but undergo dynamic turnover. Furthermore, these facts strongly support the Membrane Evolution Theory which was proposed by one of the authors in 1975.     

NITROGENASE CONFINED TO RANDOMLY DISTRIBUTED TRICHOMES IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Nitrogenase reductase (Fe-protein) was detected in the marine planktonic cyanobacterium Trichodesmium. The molecular weight was about 38 kD, as shown by western blotting using anti -Rhodospirillum rubrum nitrogenase reductase antiserum. The enzyme was confined to a limited number (ca. 10–40%) of randomly distributed trichomes in the Trichodesmium colonies, as shown by immunogold localization and transmission electron microscopy. Associated microorganisms had little or no nitrogenase. Nitrogenase showed a diel cycle in localization: present throughout the cytoplasm of cells in N₂-fixing (daytime) colonies but at the periphery of non-N₂-fixing (nighttime) colonies. This structural arrangement of N₂-fixing trichomes and nitrogenase is novel and different from the previously held paradigm for this and other diazotrophic cyanobacteria.     

H2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions

Rhodospirillum rubrum is able to produce H₂ during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H₂ production by the nitrogenase could be partially inhibited by N₂. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP chloramphenicol - DSM Deutsche Sammlung von Mikroorganismen, Göttingen - FHL formate hydrogen lyase - O.D optical density - PFL pyruvate formate lyase