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Complexed diimine in μ-N2H2[Cr(CO)5]2 undergoes a rapid H — D-exchange with deuterium ions, which is inhibited completely by acids; the rate of the H — D exchange is significantly larger for the diimine complex that for the corresponding hydrazine and ammonia complexes, which is explained by the acidic properties of the diimine protons. In the presence of catalytic amounts of strong base N2H2[Cr(CO)5]2 disproportionates fast and irreversibly to N2[Cr(CO)5]2 and N2H4[Cr(CO)5]2; a mechanism is proposed for this reaction. The reactions of the complexed diimine are compared to those of the free diimine; their significance with respect to intermediated steps of the enzymatic N2 fixation is discussed.

Dry matter accumulation, nitrogen content and N(2) fixation rates of soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to a pO(2) of 5, 10, 21, or 30% and a pCO(2) of 300 mul CO(2)/l or a pO(2) of 21% and a pCO(2) of 1200 mul CO(2)/l during the complete growth cycle were measured. Total N(2)[C(2)H(2)] fixed was increased by CO(2)/O(2) ratios greater than those in air and was decreased by ratios smaller than those in air; the effects on N(2) fixation of decreased pO(2) or elevated pCO(2) were quantitatively similar during the period of vegetative growth. Decreased pO(2) produced a smaller increase then elevated pCO(2) during the reproductive period, presumably because of the decreased sink activity of the arrested reproductive growth under subambient pO(2). At a pO(2) of 5% and a pCO(2) of 300 mul CO(2)/l total N(2) fixed was increased 125% and per cent nitrogen content in the vegetative parts was increased relative to air while that in the seed was decreased. Dry matter production was increased and reproductive growth was arrested as previously reported for plants receiving only fertilizer nitrogen. At a pO(2) of 30% and a pCO(2) of 300 mul CO(2)/l total N(2) fixed was decreased 50% and per cent nitrogen content in the vegetative part was increased relative to air while that in the reproductive structures was unaffected. Dry matter production was similarly decreased in both vegetative and reproductive structures. These effects of altered pO(2) in the aerial part on N(2) fixation are consistent with the hypothesis that the amount of photosynthate available to the nodule may be the most significant primary factor limiting N(2) fixation while sink activity of the reproductive structures may be a secondary factor.

The N2(C2H2) fixing activity was surveyed in lichens of diverse structure and habitat, all containing a blue-green phycobiont; twenty-six species from fourteen genera are reported for the first time to fix nitrogen. Rates of N2(C2H2) fixation showed great variability between different lichen samples. The proportion of heterocysts, as the assumed site of N2 fixation, was estimated in all the above lichens together with fourteen additional species. In lichens with one phycobiont, heterocysts form about 4% of the total algal cell population; in those with two phycobionts the blue-green member is found in special structures and has a very high proportion of heterocysts, typically 20-30%; the N2(C2H2) activity appears to reflect this. The high proportion may be associated with the proximity of the green phycobiont.

Successive acetylene reduction assays and legume N uptake measurements were made over a period of 23 weeks on 80 clover-rye grass pasture turfs kept in a glasshouse. Assays involved incubating the intact turfs in 5–1 containers with acetylene and measuring the Ethylene produced in 24 h. There was a close linear relationship between ethylene production and legume N uptake, with an overall correlation coefficient of 0.946; the estimated molar ratio of N2 fixed to C2H4 produced was 1:2.31. It was concluded that the acetylene reduction assay as performed provided at least a moderately accurate measure of N2 fixation in pasture turfs.

1. N2 fixation in cell-free preparations of Anabaena cylindrica was stimulated by added pyruvate. 2. A relationship between cell-free N2 fixation and decarboxylation of pyruvate was demonstrated by an almost identical response of the two processes to varying concentrations of added pyruvate. 3. Pyruvate decarboxylation was stimulated under N2-fixing conditions. 4. Decarboxylation of pyruvate was found to proceed most vigorously on particles which were most active in N2 fixation. 5. Whereas in intact cells N2 fixation was increased appreciably in the presence of CO2 in the light but decreased in the dark, in cell-free preparations N2 fixation was inhibited in the presence of CO2 in the light as well as in the dark.

The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

The same two protein components, molybdoferredoxin and azoferredoxin, are required for acetylene reduction as were shown previously to be required for N 2 fixation. Neither protein component of the N 2 -fixing system catalyzes either of these two reactions without the other. Both protein components are oxygen- and heat- (60°) sensitive and one of them, azoferredoxin, is also cold-(0°) labile. Neither molybdoferredoxin nor azoferredoxin is present in extracts of cells grown on NH 3 . No indication of the need for a third component was observed.

Semiarid desert algal crust organisms were found to fix N2 when exposed to an atomosphere which contained isotopically enriched N. Significant quantities of the N isotope were detected in the total crust N after 3 days of incubation under field-simulated conditions. Net N2 fixation rates by the algal crust organisms were 0.16 and 0.10 lb of N/acre of crust surface per day under continuous wet and cycling wet-dry conditions, respectively. Net fixation of N under field-simulated conditions adequately compensated for the removal of N by livestock. The rate of N2 fixation under field-simulated conditions increased linearly for at least 520 days. The amount of N in the algal crust was doubled during this time. No net N change was observed in dry crusts. Growing algal crusts contained 1% to 2% of the total N as extracellular NH4-N. Excretion of some fixed nitrogen was suggested by the isotopic enrichment of the extracellular N fraction and uptake of labeled N by grass seedlings (Artemesia sp.) growing on incubated crusts.

Occurrence of blue-green algae in plain land, pond side, hilly area and dry rice field soils of Chittagong University Campus and N2 fixation of some of them have been studied. Twenty four species under 20 genera were identified and their number varied from 0.11×104/g to 2.8×104/g soil. Anabaena oryzae, Calothrix sp., Cylindrospermum majus and Hapalosiphon hibernicus and Nostoc spongiaeforme were selected for their nitrogen fixing potential. Maximum amount of total nitrogen was recorded in Nostoc spongiaeforme followed by Hapalosiphon hibernicus, Cylindrospermum majus, Anabaena oryzae and Calothrix sp. after 30 days growth in batch cultures.  Key words: BGA, Distribution, Nitrogen fixation doi:10.3329/bjb.v37i1.1563 Bangladesh J. Bot. 37(1): 49-53, 2008 (June)

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

The growth constant and Y (sucrose) (grams of cells per mole of sucrose) for NH 3 -grown cultures of Clostridium pasteurianum were 1.7 times those of N 2 -grown cultures, whereas the rate of sucrose utilized per gram of cells per hour was similar for both conditions. The Y (sucrose) of chemostat cultures grown on limiting NH 3 under argon at generation times equal to those of N 2 -fixing cultures was less than that of cultures grown on excess NH 3 , but cells of NH 3 -limited cultures contained the N 2 -fixing system in high concentration. The concentration of the N 2 -fixing system in whole cells, when measured with adenosine triphosphate (ATP) nonlimiting, was more than twofold greater than the amount needed for the N 2 actually fixed. Thus, energy production from sucrose, and not the concentration of the N 2 -fixing system nor the maximal rate at which N 2 could be fixed, was the limiting factor for growth of N 2 -fixing cells. Either NH 3 or some product of NH 3 metabolism partially regulated the rate of sucrose metabolism since, when cultures fixing N 2 , growing on NH 3 , or growing on limiting NH 3 in the absence of N 2 were deprived of their nitrogen source, the rate of sucrose catabplism decreased. Calculations showed that the rate of ATP production was the growth rate-limiting factor in cells grown on N 2 , and that the increased sucrose requirement of N 2 -fixing cultures in part reflected the energy demand of N 2 fixation. Calculations indicated that whole cells require about 20 moles of ATP for the fixation of 1 mole of N 2 to 2 moles of NH 3 .

Research in the biochemistry and biophysics of N2 fixation has progressed along two separate but related lines. One deals mainly with the catalytic activity of the enzyme nitrogenase, while the other is concerned chiefly with its physical and chemical characteristics. Productive research in the former was initiated with the preparation of active cell-free extracts in 1960, but characterization work did not get well under way until into the mid-1960’s. By then the over-all reaction characteristics had become reasonably clear, though the detailed reaction mechanism was not established, nor is it yet. Since the mid-1960’s, understanding of the nitrogenase reaction has become increasingly refined, and major advances in defining the physical-chemical nature of the enzyme have been made possible with the availability of pure nitrogenase proteins for analysis. Until now it has been feasible and also fruitful to pursue these two areas rather independently; however, as experimentation becomes more definitive and bears more directly on the mechanism of nitrogenase action, these areas seem destined to overlap increasingly as they blend toward the ultimate goal, the description of nitrogenase catalysis in terms of molecular structure.

Hydrazine and hydroxylamine are highly inhibitory to the fixation of N2 by cell-free extracts from Clostridium pasterianum. After cell-free extracts from C. pasteurianum had fixed 15N2 for short periods, hydrazine (as its benzalazine derivative) and hydroxylamine (as the copper salt of pyruvic oxime), were recovered with the aid of carrier hydrazine or hydroxylamine. Although the ammonia had concentrations of 15N as great as 32 atom% excess, the benzalazine and pyruvic oxime had no significant 15N excess. Hydrolysis to liberate possible bound forms of hydrazine and hydroxylamine did not alter these results. The data give no support to the concept that hydrazine or hydroxylamine serves as an intermediate in biological N2 fixation.

The aim of this chapter is not to review exhaustively the physiology of the free-living N2-fixing microbes. Such a task would require a book to itself and the information is already covered more than adequately in Wilson’s (1958) review, Rubenchik’s (1960) monograph on azotobacters and the encyclopaedic work of Mishustin and Shi’nikova (1966). Jensen (1965) has touched on many features of their physiology in an exhaustive account of their ecology. My present aim is to relate the advances over the last decade in our knowledge of the chemistry and enzymology of the N2 fixation process to the general physiology of free-living N2-fixing microbes; in general I shall take the information in Wilson’s (1958) review for granted.

N2 fixation is accomplished by the combined action of two proteins, and this complicates the problem of nomenclature. The problem is particularly acute because it is not clear whether the proteins act independently or as a complex, nor is it clear what specific action each protein effects. Nitrogenase or the N2-fixing complex refers to the two proteins acting together. The individual proteins of nitrogenase will be termed the Mo-Fe protein and the Fe protein; some workers refer to these as ‘protein 1’ or ‘fraction 1’ and ‘protein 2’ or ‘fraction 2’ respectively. Although these designations are not completely definitive, because other Fe proteins do exist in the N2-fixing cells, they seem preferable currently to azoferredoxin and molybdoferredoxin. Mortenson, Morris and Jeng (1967) introduced the term azoferredoxin to designate the N2-binding protein of the nitrogenase complex, but later work (Bui and Mortenson, 1968) suggested that it is the Mo-Fe protein which binds N2. When the chemical nature of the Mo-Fe and Fe proteins and their functions are better established a more rational nomenclature can be assigned.

SOME termites can live on a diet of cellulose filter paper1. This characteristic prompted Hungate2 to question the possibility of N2 fixation in termites by estimating the N balance of growing termite colonies, but he found no net increase in total colony N. Toth3, on the other hand, incubated macerated termites in an N-free organic medium and observed an increase in fixed N in the solution. He used long incubation times, however, and possibly selected for N2-fixing organisms in the medium. We decided to reinvestigate the question of N2 fixation in termites by exploiting the sensitivity of the acetylene reduction assay, a reliable indicator of N2 fixation4,5. Preliminary reports of these results have already been published6,7.

Lichens with N2-fixing blue-green algae as phycobionts or cephalodia have the ability to fix N2 from the air. This was demonstrated by using 15N-enriched nitrogen in laboratory experiments (Bond and Scott 1955, Watanabe and Kiohara 1963, Rogers et al. 1966, Millbank and Kershaw 1969) and in situ (Fogg and Stewart 1968). The present investigation gives further information about the extent of N2 fixation occurring in phycobionts and also about the ability of the lichens to resume N2 fixation after varied periods of drought. The determinations of the capacity of N2 fixation are carried out by using the acetylene reduction technique. Most lichens are very resistant to desiccation. They rapidly recover full activity of photosynthesis and respiration when moistened after a prolonged dry period. The length of the dry period which the lichens can endure, depends upon several factors. One of them is the nutritional state before desiccation, another the conditions of storage. In addition, the environmental conditions in which the lichens are growing influence their ability of surviving long dry periods. Generally, the lichens can withstand dry periods far longer than what they

Arctic lichens are photosynthetically well adapted to low temperatures. In Stereocaulon paschale (L.) Fr. and Nephroma arcticum (L.) Torrs. the optimal temperature for net photosynthesis is between 0?C and 5?C. But the optimal temperature for N2 fixation is about 15?C and at 0?C the lichens are inactive. Therefore N2 fixation is not well adapted to average arctic conditions and this may account for the low nitrogen content of arctic soils. The significance of local climatic differences for nitrogen supply is discussed.