Nitrogenase Activity and Photosynthesis in Plectonema boryanum

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.     

Nitrogen fixation by Anabaena cylindrica

Summary The effects of oxygen, light and photosynthesis inhibitors on nitrogenase activities in Anabaena cylindrica batch cultures were followed as a function of time after inoculation. During the early rapid growth period the nitrogenase activities of cultures grown under air/CO₂ or N₂/CO₂ were relatively resistant to oxygen and DCMU inhibition. These cultures also exhibited oxygen-dependent nitrogenase activity in the dark of up to 50% of that measured in the light. After active growth ceased the cultures continued to slowly grow for a prolonged period of time. The nitrogenase activities of these old cultures were very sensitive to oxygen and DCMU inhibition. These cultures also had little or no dark nitrogenase activities. The photosynthesis inhibitor DBMIB was not a specific inhibitor of light-driven electron transport since it inhibited both light and dark nitrogenase activities. Nitrogenase activities induced under oxygen-free/CO₂ gas mixtures initially were significantly more sensitive to oxygen inhibition than those induced under air/CO₂. We discuss these results in relation to heterocyst function.     

Oxidation of diaminobenzidine in the heterocysts ofAnabaena cylindrica

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the honeycomb region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H₂O₂, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O₂-labile nitrogenase.     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.     

Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria): Temperature Characteristics and Potential Use in Biophotolysis

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10⁴ ergs cm⁻² s⁻¹. Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of M. laminosus NZ-86-m produced hydrogen at comparable rates to Anabaena cylindrica. The conversion efficiency of light to hydrogen energy at maximum rates of hydrogen production was 2.7%.     

The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Evidence has been obtained that NADPH may serve as a physiological source of reducing power for nitrogenase activity in Azotobacter vinelandii. NADH was ineffective. Electron transfer from NADPH to nitrogenase depended on four factors native to A. vinelandii cells: azotobacter ferredoxin, azotoflavin, a component replaceable by spinach ferredoxin-NADP⁺ reductase and another soluble, heat-labile component not yet chemically characterized. The four factors probably constitute an electron transport chain between NADPH and nitrogenase.     

Hydrogenase activity in the thermophile Mastigocladus laminosus

Hydrogenase activity in the thermophilic cyanobacterium, $\text{Mastigocladus}$$\text{laminosus}$ was studied both $\text{in vivo}$ and $\text{in vivo}$ hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H₂-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O₂-independent reaction. $\text{In vitro}$ hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for $\text{Anabaena cylindrica}$.     

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O₂ production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.     

Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O₂ for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O₂ tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.