Nitrogen fixation and heterocysts in the blue-green alga Anabaena cylindrica

The site of nitrogen fixation in the blue-green alga Anabaenacylindrica Lemra (Fogg strain) was investigated. Less than 4%of the total nitrogen fixed during a relatively short period(5-15 min) was recovered in heterocysts. When estimated on thecellular nitrogen basis, vegetative cells can fix molecularnitrogen at the same rate as do heterocysts. There was no positivecorrelation between nitrogen fixation and heterocyst formation.Results do not support the hypothesis that the heterocyst isthe main site for nitrogen fixation in blue-green algae. ¹ This work was supported by grant (No. 38814) from the Ministryof Education. (Received July 23, 1971; )     

The Effect of Available Nitrate and Ammonium-Nitrogen on the Growth of Two Nitrogen-Fixing Blue-Green Algae

The effect of combined nitrogen supplied as nitrate or ammonium-nitrogenon the growth of two nitrogen-fixing blue-green algae, Nostocentophytum and Calothrix scopulorum, has been studied. Thesespecies have been isolated from marine environments. Both algaegrew as vigorously on elemental nitrogen as in the presenceof combined nitrogen. Growth was equal at all levels of nitrate-nitrogenemployed but high levels of ammonium-nitrogen proved inhibitoryor even toxic to the algae. Nostoc was slightly more susceptibleto high ammonium-nitrogen levels than was Calothrix. Increasein pH of the medium from 7.2 to 8.4 increased the toxic effectof ammonium-nitrogen although relative growth at the variouslevels of nitrate-nitrogen was not affected. The results suggestthat the different effects of ammonium-nitrogen on the growthof freshwater and marine blue-green algae may be due in partat least to the different pH levels of freshwater and marineenvironments in which the algae grow, rather than to any inherentdifference between the two groups.     

Algal nitrogen fixation in the tropics

Summary a)Nitrogen fixation in rice fields. Nitrogen-fixing blue-green algae grow abundantly in tropical regions and are particularly common in paddy fields. Their possible role in the nitrogen accumulation of soil has been studied. The most vigorous nitrogen-fixing blue-green algae have been assessed for use as green manure in rice fields and favorable effects have been reported in India and other countries. b)Nitrogen fixation by algae in water. The planktonic blue-green algae occur abundantly at certain time of the year in sea water and lake water, and some of them are known to be nitrogen fixers. Certain Japanese species of blue-green algae can withstand high temperatures including ten nitrogen-fixing species from hot-spring waters. c)Nitrogen fixation by symbiotic blue-green algae. Certain species of blue-green algae form associations with other organisms such as fungi, liverworts, ferns and seed plants. The relationship between these two organisms is on one occasion commensal and on others symbiotic. Certain symbiotic blue-green algae are provided with the ability to fix the atmospheric nitrogen.     

Algal nitrogen fixation on the lava field of Heimaey,Iceland

Summary Nitrogen fixation (C₂H₂ reduction) by blue-green algae occurring on the juvenile lava field of Heimaey, Iceland was examined both in the laboratory (potential at 20° C and 39° C) and in the field, three and a half years after the volcanic eruption.Already at this early stage of colonization representatives of unicellular and filamentous heterocystous and non-heterocystous blue-green algae were commonly observed. The predominating algae were Nostoc sp. (20° C) and Schizothrix sp. — Microcoleus chthonoplastes, (39° C), the former often in association with the protonemata-rhizoids of moss plants.The potential for nitrogen fixation was recorded at an average rate of 109.2 (20° C) and 138.1 (39° C) ng N g^-1 h^-1 in soil collected from localities randomly distributed over the lava field.Tests for nitrogen fixation performed in situ revealed significant fixation activities in all the eleven localities subject to examination. The activities ranged from 2.8 to 63.4 (mean 21.5) ng N g^-1 h^-1 and 1.9 to 17.7 (mean 7.9) ng N cm^-2 h^-1.All the nitrogen fixation data noted imply that blue-green algae contribute a substantial part of the nitrogen input to the lava. Further, it was found that material incubated under micro-aerophilic conditions exhibited considerably enhanced nitrogenase activity.The role of nitrogen-fixing blue-green algae in general and Nostoc muscorum in particular in being suitable as pioneering organisms preparing the bare lava for ingress of other plants is also discussed.     

Cyanidium caldarium as a bridge alga between Cyanophyceae and Rhodophyceae: Evidence from immunodiffusion studies

The primer-independent phosphorylase isozyme, a₂, of Cyanidiumcaldarium was used for immunization of rabbits. The immune serumwas tested against pure a₂ isozymes from blue-green, red, andgreen algae. Double immunodiffusion in agar indicated that therewas structural similarity in the isozyme from Cyanidium caldarium,the blue-green algae, Oscillaloria princeps, Pleclonema nostocorumand the red alga, Rhodymenia pertusa. Complete fusion of theprecipitin lines was obtained with these algae. However, onlypartial fusion was observed with the a₂ isozymes from Chlorophyceaesuch as Chlorella pyrenoidosa and Spirogyra setiformis. Spurformation on the precipitin lines occurred when the isozymesfrom these algae were tested against the immuneserum. The results were interpreted as indicative of the possible transitionstatus of Cyanidium caldarium between prokaryotic blue-greenalgae and eukaryotic red algae. It would appear that the Chlorophyceaeevolved along different lines from Cyanophyceae than did theRhodophyceae. (Received November 25, 1975; )     

Nitrogen fixation in lakes

Summary Determinations in the open waters of lakes using N¹⁵ as a tracer show that nitrogen fixation is generally associated with the presence of heterocystous blue-green algae and is light dependent. Although nitrogen-fixing blue-green algae tend to be abundant when the concentration of nitrate or ammonia in the water is low, fixation itself is not necessarily inhibited by the presence of these sources of combined nitrogen. The activity of nitrogen-fixing blue-green algae shows a direct relationship to concentration of dissolved organic nitrogen. As a result of the interaction of such factors, nitrogen fixation per unit area of lake surface per year tends to be greatest at an early stage of eutrophication. In relation to the total nitrogen budget of a lake the contribution of biological nitrogen fixation to the income is probably always small but at certain times and in particular water strata it may contribute a major part of the nitrogen assimilated by the phytoplankton.     

Nitrogen Turnover in Marine and Brackish Habitats II. Use of 15N in Measuring Nitrogen Fixation in the Field

A technique for measuring nitrogen fixation in situ by naturalpopulations of blue-green algae is described. It involves exposingtest samples of known area to a gas mixture containing molecularnitrogen enriched with ¹⁵N and measuring the rate of incorporationof the isotope over a standard 24-h exposure period. The accuracyof the method is not seriously affected by changes in pH, pCO²and humidity which may occur during the exposure period, orby the degassing procedure used to remove air from the exposureflask prior to introduction of the isotope. Temperature andpN² values inside the exposure flask are different from thoseto which natural populations are exposed outside, and correctionsfor these have to be made in calculating the final results.The minimum pN² which allows optimum fixation by Calothrix scopulorumin the presence of 0.2 atm. oxygen is 0.4 atm. In an area ofthe supra-littoral fringe of a rocky shore and in an area ofsand dune-slack over a 12-month period nitrogen fixation ishigh in spring and autumn and negligible in winter. On the rockyshore fixation is low in summer; in the dune-slack summer nitrogenfixation is erratic. Nitrogen-fixing efficiency in terms ofnitrogen fixed per unit weight of test material is high whenalgal recolonization is occurring. The mean fixation rate perannum corresponds to approximately 2.5 g/m2/annum for the rockyshore. The nitrogen fixed per annum represents approximately41 per cent and 21 per cent of the mean total nitrogen presentper annum on the rocky shore and in open areas of the sand dune-slackrespectively.     

Rapid physiological assays for nutrient demand by the plankton. I. Nitrogen

Three assays for nitrogen demand were compared on samples ofnatural plankton and on green and blue-green algal species inculture. The most reliable guide was selective luxury uptakeof nitrogen by the plankton after enrichment with a 10-to-1(wt/wt) mixture of inorganic N and P. Ammonium transport capacity,measured either directly (cultures) or by V_max for uptake ofthe NH₄⁺ analogue methylammonium (lakewaters), was generallyhigh in lakewaters with low dissolved inorganic N-to-P ratiosand in N-deficient cultures of green algae. By contrast, transportcapacity was much reduced in both natural and cultured populationsof heterocystous blue-green algae growing under conditions oflow combined inorganic nitrogen. Ammonium enhancement assays(heterotrophic CO₂, fixation after NH₄⁺ enrichment) were conductedat monthly intervals on eutrophic Lake Rotorua. There was astrong, negative correlation between this indicator of N deficiencyand dissolved inorganic N-to-P ratios below a threshold of 6:1.Ammonium enhancement was not, however, a reliable indicatorof combined inorganic N-demand by populations of heterocystousblue-green algae. All three assays provided strong evidenceof a persistent shortage of nitrogen relative to phosphorusfor algal production in Lake Rotorua.     

Resistance to blue-green algal toxins by Bosmina longirostris

Bosmina longirostris was resistant to strains of Microcysrtsaeruginosa and Anabaena flosaquae previously reported to havelethal toxic effects on cladocerans. These blue-green algaewere of poor nutritional value to B.longirostris; survivorshipwas increased when fed these algae but reproduction was negligible.Results of feeding selectivity experiments showed that B.longirostrisdid not avoid consuming these blue-green algae, indicating thatthe mechanism for resistance must be post-ingestion. These resultssuggest that, unlike the other cladocerans tested, B.longirostriscould potentially coexist with toxic blue-green algal blooms.     

Quantitative relationship between two reaction centersin the photosynthetic system of blue-green algae

The quantitative relationship between reaction centers I andII was studied with blue-green algae Anabaena cylindrica, Anabaenavariabilis and Anacystis nidulans grown under different lightconditions. The number of reaction centers I was estimated fromthe P700 content and that of reaction centers II, from the O₂yield of repetitive short flashes. Supplementary determinationswere done with three other blue-green algae and one red alga.The maximum number of reaction centers II counted from the O₂yield of repetitive short flashes was markedly smaller thanthe total number of P700 in all algae tested when the algaewere grown under weak light; in the extreme case (Anabaena cylindrica),the ratio was only 0.258?0.015. This ratio became larger andclose to unity when the algae were grown under stronger light.Variation in the number of reaction centers in a single cellsuggested that reaction center I was a variable component. Ourresults indicate that the proportion of the two reaction centersmay markedly vary in blue-green algae depending on the growthconditions (Received November 13, 1978; )     

The Relation of Acetylene Reduction to Heterocyst Frequency in Blue-Green Algae

The variation of acetylene reduction activity and heterocystfrequency with culture age, was studied in five species of blue-greenalgae. The heterocyst frequency varied between 2.5 to 12.3 percent of total cells; and the acetylene reduction activity varied2.0-fold (average). It is suggested that an estimate of thenitrogen fixation rates in blue-green algae may be made by insitu heterocyst counts and acetylene reduction measurements.     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

The Effect of Temperature on Nitrogenase Activity

Acetylene reduction by detached nodules of four non-legumes(Alnus, Hippopha, Myrica, Casuarina), five legumes (Glycine,Lupinus, Pisum, Vicia, Medicago), and two blue-green algae (Anabaena,Plectonema) was tested with respect to the effect of temperatureon nitrogenase activity. In all cases the activity was sensitiveto temperature change, and with the exception of the legumesthere was a simple exponential response to temperature up tothe optimum. The temperature sensitivity of nitrogenase activityin the two blue-green algae was reduced in low light intensities.Temperature data for several other species are compared, anda simple method of correcting for temperature differences suggested.It is emphasized that allowance for the sensitivity of acetylenereduction to temperature differences must be made if field dataare to be used for purposes of comparison.     

ADAPTIVE FORMATION OF NITRATE REDUCING SYSTEM IN ANABAENA CYLINDRICA

1. Changes in capacities for reducing nitrate, nitrite and hydroxylaminecaused by provision or depletion of various nitrogen sourceswere investigated with a nitrogen fixing blue-green alga, Anabaenacylindrica, and adaptive nature of these reducing system wasdemonstrated.
2. It was found that, under light-aerobic conditions,nitrate-and nitrite- reducing systems were induced by nitrateor nitritebut not by N₂ ammonia and glutamate. On the otherhand, theactivity of enzymes pertaining to hydroxylamine reductionwasstimulated equally by nitrate, nitrite and N₂. The latteractivitywas suppressed markedly in the presence of ammoniaor glutamate.
3. Adaptive formation of nitrite reducing systemis completelyinhibited by chloramphenicol, a potent inhibitorof proteinsynthesis. No formation was also observed under theanaerobiccondition or in the dark.
4. On the basis of thesefindings, a tentative scheme for pathwaysof nitrate reductionand nitrogen fixation in Analaena cylindricawas proposed.

(Received August 22, 1962; )     

Nitrogen fixation by the unicellular blue-green alga Aphanothece

Summary Frequent growth of unicellular blue-green alga Aphanothece sp. was observed in medium free of combined nitrogen. Its generation time was 12 h and more than 2 mg of nitrogen was fixed in 25 days. Its growth and nitrogen fixation were comparable to other heterocystous algae.     

Deep placement: A method of nitrogen fertilizer application compatible with algal nitrogen fixation in wetland rice soils

Summary The effect of different methods of nitrogen fertilizer application on the algal flora and biological nitrogen fixation (Acetylene-reducing activity) in a wetland rice soil was studied in pot and field experiments. Broadcast application of urea inhibited nitrogen fixation and favored the growth of green algae. In contrast, deep placement of urea supergranules (1–2 g urea granules) did not suppress the growth of N₂-fixing blue-green algae and permitted acetylene-reducing activity on the soil surface to continue virtually uninhibited.     

Nitrogen fixation (acetylene reduction) associated with communities of heterocystous and non-heterocystous blue-green algae in mangrove forests of Sinai

Summary High rates of nitrogen fixation (acetylene reduction) are associated with communities of heterocystous and non-heterocystous blue-green algae, which are widespread and abundant in the coastal mangrove forests of the Sinai Peninsula.Heterocystous forms, particularly representatives of the Rivulariaceae, grow in aerobic environments, where nitrogenase activity may be limited by the availability of nutrients such as Fe and PO₄–P. Desiccated communities of Scytonema sp. reduce acetylene within ten minutes of wetting by tidal sea water. Communities dominated by the non-heterocystous Hydrocoleus sp., Hyella balani, Lyngbya aestuarii, Phormidium sp. and Schizothrix sp., occur in close contact with anaerobic sediments and reduce acetylene in the dark as well as in the light.Nitrogen fixation in all these communities is light dependant and may be supplemented by an alternative source of reductant in the dark. The indications are that nitrogen fixation by these communities of blue-green algae, makes a significant contribution to the overall nitrogen input of the mangrove ecosystem.     

Optimization of carbon dioxide and nitrogen fixation by the blue-green alga Anabaena in freshwater blooms

Summary In the bloom-forming filamentous blue-green algae Anabaena, both carbon dioxide and nitrogen fixation share a dependence on light. During daylight, A. reduces direct competition between these processes for light-generated reductant by optimizing carbon fixation during late morning hours while optimizing nitrogen fixation during afternoon hours.Sequential optimization was examined from both biochemical and ecological perspectives. Biochemically, it is sound to optimize carbon prior to nitrogen fixation, due to the higher sensitivity of the former to afternoon increases in dissolved oxygen levels which commonly occur in blooms. It is also advantageous to first assure adequate supplies of fixed carbon prior to incorporating fixed nitrogen. Ecologically, the sequence represents optimal use of radiant energy. A. is able to shift energy flow from a highly inhibited to a less inhibited process, thereby maintaining a sink for photo-generated reductant. Both A. and a non-nitrogen fixing diatom community show similar carbon fixation efficiencies during morning and midday hours. During afternoon however, A. diverts significant portions of photo-reductant from carbon to nitrogen fixation. This allows A. to optimize carbon fixation while maintaining access to nitrogen during periods of ambient nitrogen shortage. Accordingly, A. blooms usually appear during summer months when both nitrogen starvation and photosynthetically-active radiation inputs are maximal.     

Does N2 fixation meet the nitrogen requirements of heterocystous blue-green algae in shallow eutrophic lakes?

Summary Phytoplankton net carbon uptake and nitrogen fixation were studied in two shallow, eutrophic lakes in South Sweden. Ranges of diurnal net carbon uptake were estimated by subtracting 24-h respiration rates corresponding to 5–20% of P _max, respectively, from daytime carbon uptake values. total nitrogen requirement of the phytoplankton assemblage was determined from the diurnal net carbon uptake, assuming a phytoplankton C:N ratio of 9.5:1. Nitrogen supplied by nitrogen fixation only occasionally corresponded to the demands of the total phytoplankton assemblage. When heterocystous algae made up a substantial proportion (10%) of the total phytoplankton biomass, nitrogen fixation could meet the requirements of heterocystous blue-green algae on c. 50% of the sampling occasions. Nitrogen deficiencies in heterocystous algae were most probably balanced by the simultaneous or sequential assimilation of dissolved inorganic nitrogen. It was concluded that uptake of ammonium or nitrate, regenerated from lake seston and sediment, is the main process by which growth of phytoplankton is maintained during summer in the lake ecosystems studied.     

Growth and Nitrogen Fixation by Westiellopsis prolifica Janet

The blue-green alga Westiellopsis prolifica Janet has been isolatedin unialgal bacteria-free culture. Evidence of the ability ofthis algs to fix elementary nitrogen was obtained by demonstrationof increase in total combined nitrogen in cultures by the micro-Kjeldahlmethod and also by the uptake of ¹⁵N as demonstrated by theuse of the mass spectrometer. The growth and nitrogen-fixingcapacity of the alga was studied in relation to temperatureand light intensity. Increase in dry weight was greatest at40° C but the optimum for nitrogen fixation was between30 and 35°C. A considerable proportion of the nitrogen fixedwas released in soluble organic form into the medium.