Nitrogen Fixation (Acetylene Reduction) by Epiphytes of Freshwater Macrophytes

The involvement of epiphytic microorganisms in nitrogen fixation was investigated in a shallow freshwater pond near Ithaca, N.Y. The acetylene reduction technique was used to follow diel and seasonal cycles of nitrogen fixation by epiphytes of Myriophyllum spicatum. Acetylene-reducing activity was maximal between noon and 6 p.m., but substantial levels of activity relative to daytime rates continued through the night. Experiments with the seasonal course of activity showed a gradual decline during the autumn months and no activity in January or February. Activity commenced in May, with an abrupt increase to levels between 0.45 and 0.95 nmol of ethylene formed per mg (dry weight) of plant per h. Through most of the summer months, mean rates of acetylene reduction remained between 0.15 and 0.60 nmol/mg (dry weight) per h. It was calculated from diel and seasonal cycles that, in the pond areas studied, epiphytes were capable of adding from 7.5 to 12.5 μg of N per mg of plant per year to the pond. This amount is significant relative to the total amount of nitrogen incorporated into the plant. Blue-green algae (cyanobacteria), particularly Gloeotrichia, appeared to bear prime responsibility for nitrogen fixation, but photosynthetic bacteria of the genus Rhodopseudomonas were isolated from M. spicatum and shown to support high rates of acetylene reduction.     

Nitrogen fixation in lake Mendota, 1971–1973

The contribution of nitrogen fixation to the nitrogen budget of Lake Mendota has been calculated. On average, the equivalent of 1.28 × 10⁵ kg of NH₃ (as determined by the acetylene reduction technique) was added to this eutrophic lake during June, July and August. Diurnal variation (approximately two-thirds of the day's fixation occurs prior to noon) in algal nitrogen fixation, and variation of fixation with depth (3.6% of the fixation in the column occurs in the top decimeter) were characterized as prerequisites to this calculation.     

Nitrogen cycling in an Antarctic ecosystem 2. Estimation of the amount of nitrogen fixation in a moss community on East Ongul Island

Nitrogen fixation by cyanobacteria in a moss community on East Ongul Island (69°00'S 39°35'E), Antarctica was investigated using the acetylene reduction method. The mean acetylene reduction rate at 10°C and 200 μE·m⁻²·s⁻¹ photosynthetically active radiation was 7.12 nmol C₂H₄ per square centimeter of moss community per hour. The effects of temperature, radiation, desiccation and rehydration on the acetylene reduction rates were examined. A simple predictive model was constructed in order to estimate the amount of nitrogen fixed in the field. Using this model, the daily amount of nitrogen fixation was calculated from microclimatic data (temperature and radiation) measured in the experimental field at Syowa Station on East Ongul Island between 1983 and 1984. The cumulative amount of nitrogen fixation in the growing season during this period was estimated to be 329 mg N per square meter of moss community. It is suggested that nitrogen fixation by cyanobacteria in the moss community is important as a nitrogen source for the community growth on East Ongul Island.     

The Effects of Moisture on Acetylene Reduction by Mats of Blue-green Algae in Sub-tropical Grassland

During the dry winter months desiccation is the main factorpreventing nitrogen fixation by mats of blue-green algae inkikuyu lawns. In the wetter summer months a few days of sunshinewithout rain will dry out the algal mats and depress nitrogenfixation unless the algae are protected by swards of grass.Dry mats resume nitrogen fixation within an hour of becomingdamp and attain their normal rate of fixation within 5 h. Maximumfixation rates occur at 100 per cent relative humidity and withsoil moisture contents of 22 to 42 per cent. Dry algal matsare unable to take up sufficient water as vapour (even in saturatingconditions) to permit resumption of acetylene reduction. Algal-dominatedsoils show greater water retention than adjacent bare soils.     

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.     

A NEW NITROGEN-FIXING NON-LEGUME: CHAMAEBATIA FOLIOLOSA (ROSACEAE)

Specimens of Chamaebatia foliolosa Benth. with nodule structures on their roots fix atmospheric nitrogen. The nodules are similar to those of other non-legumes in gross morphology and structure, containing hyphal strands, some with club-shaped vesicles at their ends. A fixation rate of 130 nmoles N₂ per g fresh weight per hr is reported by using ¹⁵N₂ as a tracer. Equivalent rates of acetylene reduction were observed.     

Diurnal changes in maize leaf photosynthesis : I. Carbon exchange rate, assimilate export rate, and enzyme activities

Diurnal changes in photosynthetic parameters and enzyme activities were characterized in greenhouse grown maize plants (Zea mays L. cv Pioneer 3184). Rates of net photosynthesis and assimilate export were highest at midday, coincident with maximum irradiance. During the day, assimilate export accounted for about 80% of net carbon fixation, and the maximum export rate (35 milligrams CH₂O per square decimeter per hour) was substantially higher than the relatively constant rate maintained through the night (5 milligrams CH₂O per square decimeter per hour). Activities of sucrose phosphate synthase and NADP-malate dehydrogenase showed pronounced diurnal fluctuations; maximum enzyme activities were generally coincident with highest light intensity. Reciprocal light/dark transfers of plants throughout the diurnal cycle revealed that both enzymes were deactivated by 30 minutes of darkness during the day, and they could both be substantially activated by 30 minutes of illumination at night. During 24 hours of extended darkness, sucrose phosphate synthase activity declined progressively to an almost undetectable level, but was activated after 1.5 hours of illumination. Thus, the diurnal fluctuation in maize sucrose phosphate synthase can be explained by some form of light modulation of enzyme activity and is not due to an endogenous rhythm in activity. No diurnal fluctuations were observed in the activities of NADP-malic enzyme or fructose 6-phosphate-2-kinase. Phosphoenolpyruvate carboxylase was activated by light to some extent (about 50%) when activity was measured under suboptimal conditions in vitro. The results suggested that the rates of sucrose formation and assimilate export were closely aligned with the rate of carbon fixation and the activation state of sucrose phosphate synthase.     

Role of nitrogen assimilation in seed development of soybean

A nondestructive acetylene reduction assay for nitrogenase activity of soybean (Glycine max L. Merr) field plots is presented. Plots consisted of 120 × 150 × 30 centimeter boxes containing 65 plants. The plants were grown in a medium grade sand under controlled nutrient, moisture, and root temperature conditions. Acetylene at a concentration of 10 milliliters per liter was circulated through manifolds in the chambers; equilibration required 5 minutes, and activity was linear with time. Optimum growth and assay environments resulted in activity of 70 micromoles ethylene per plant per hour. Plant development and yield were comparable to soil-grown companion plots.

The well accepted hypothesis that developing seeds deprive the nodules of carbohydrate was not substantiated. The nondestructive acetylene reduction profile did not decline until 30 days after the onset of seed development (R-5). This result was consistent with reports from the literature which indicated that 60% of seasonal nitrogen was fixed after R-5. Further, a high correlation shown between integrated seasonal acetylene reduction and yield (r = 0.999) suggested a cooperative relationship between the roots and shoot. A reduction in source:sink ratio (60% defoliation) after R-5 had no effect on acetylene reduction. This showed that neither an increase in sink demand by the pods nor a carbon shortage during podfill decreased dinitrogen fixation. A conceptual model relating seed growth with carbon and nitrogen assimilation is proposed.

     

Algal and bacterial non-symbiotic nitrogen fixation in paddy soils

Summary The nitrogen fixing activity of three Ivory Coast soils was tested in the laboratory by the acetylene reduction assay and the Kjeldahl method. Nitrogen fixation due to algae was estimated to be of the order of 4 to 8 (acetylene method) and 7 μg N per g soil per day (Kjeldahl method). Nitrogen fixation due to bacterial activity in the rice rhizosphere was estimated to be of the order of 2 to 5 (acetylene method) and 1 to 3 μg N per g soil per day (Kjeldahl method). These results emphasize the importance of the bacterial nitrogen fixation in the rhizosphere which had been hitherto overlooked. Comparison of acetylene method and Kjeldahl method results shows discrepancies the origin of which has been discussed. Time course of acetylene reduction by rhizosphere soils exhibits a lag phase which may be attributed to Postgate's switch off — switch on process.     

Diel Nitrogen Fixation by Cyanobacterial Surface Blooms in Sanctuary Lake, Pennsylvania

Diel nitrogen fixation studies were conducted with assemblages of cyanobacteria sampled from surface blooms on Sanctuary Lake, Pa. The studies were conducted between July and September of 1982 to 1985 by using the acetylene reduction technique. Assemblages with the lowest cell concentrations (0.9 × 10⁹ to 1.0 × 10⁹ cells per liter) exhibited nitrogen fixation activity throughout the day, with maximum fixation rates occurring in mid to late afternoon; fixation proceeded throughout the night at rates equivalent to 23 to 28% of the afternoon maximum. In studies conducted with the highest cell concentrations (3.7 × 10⁹ to 6.7 × 10⁹ cells per liter), fixation rates reached maximum values in mid to late morning. The rates declined rapidly throughout the midday period and subsequently ceased from late afternoon until sunrise on the following day. The afternoon decline and cessation of fixation exhibited by high cell concentrations correlated with photosynthetically induced low total CO₂ and supersaturating O₂ concentrations. The midday decline could be prevented and partially reversed by experimentally lowering O₂ and increasing total CO₂ concentrations. Under experimental conditions which simultaneously prevented supersaturating O₂ concentrations and maintained high total CO₂ availability, nitrogen fixation continued throughout the solar day, with maximum rates occurring at midday. These observations indicate that temporal changes in photosynthetic activity may affect diel fluctuations in nitrogen fixation.     

Diurnal variation in n(2) fixation and photosynthesis by aquatic blue-green algae

Rates of ¹⁴CO₂ fixation, O₂ evolution, and N₂ fixation (acetylene reduction) by natural populations of blue-green algae recovered from Lake Mendota were measured at frequent intervals between sunrise and sunset. Photosynthesis and N₂ fixation were depressed during midday when light intensity was greatest. As the light intensity rose, most of the algal population migrated to deeper, light-limited waters where radiation damage would be diminished. As the relative rate of N₂ fixation compared to CO₂ fixation increases with depth, it is suggested that the algae maintain balanced growth by migrating vertically via buoyancy regulation. High concentrations of dissolved O₂ in lake water may inhibit N₂ fixation by enhancing photorespiration. Several factors such as photosynthetic rate, light intensity, dissolved O₂, species composition, and vertical and horizontal migration all affect observed rates of in situ N₂ fixation.     

Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids

In vivo CO₂ fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.

Forage harvest and nitrate application reduced both in vivo and in vitro CO₂ fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.

The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.

The results demonstrate that CO₂ fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO₂ fixation for attached and detached alfalfa nodules at low CO₂ concentrations (0.13-0.38% CO₂) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO₂ fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

     

Photosynthesis and photosynthate partitioning in n(2)-fixing soybeans

Leaf area, chlorophyll content, net CO₂ photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. A second set (Nod+/−) was inoculated and had N₂ fixation as its sole source of nitrogen. A third group (Nod⁻) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. The mean net micromole CO₂ uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/− test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/− utilized their chlorophyll more efficiently for photosynthetic CO₂ uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/− but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/− sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation.     

In situ acetylene-ethylene assay of biological nitrogen fixation in lowland rice soils

Summary An in situ device for assaying biological nitrogen fixation in flooded rice soils, using the acetylene reduction method, was developed. Diurnal variations in acetylene reduction by an inoculated field plot and by laboratory-grown cultures of nitrogen-fixing algae showed a prominent single-peak pattern of nitrogenase activity. The peak occurred at mid-day for laboratory-grown algae and at late afternoon for the algae grown in the field plot. Some nitrogenase activity was noted during the night. Acetylene reduction studies in rice fields of Albay province, Philippines, showed an estimated fixation of 18.5 to 33.3 kg N/ha each cropping season for the fields of Puro soil and 2.3 to 5.7 kg N/ha each cropping season for the fields of Santo Domingo soil. re]19751202     

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.     

The Azolla-Anabaena azollae relationship

The heterocystous blue-green alga, Anabaena azollae, was isolated from the leaf cavities of the water fern, Azolla caroliniana, where it occurs as an endophyte. The isolated alga was capable of light dependent CO₂ fixation and acetylene reduction. Aerobic dark acetylene reduction occurred and was dependent upon endogenous substrates. Vegetative cells of the alga reduced nitro-blue tetrazolium chloride (NBT) to blue formazan. Heterocysts did not. Heterocysts reduced triphenyl tetrazolium chloride (TTC) to red formazan faster than vegetative cells. Reduction of TTC by both heterocysts and vegetative cells was much more rapid than has been reported for free-living heterocystous blue-green algae. Both NBT and TTC inhibited acetylene reduction and CO₂ fixation. The inhibition by TTC was more closely correlated to the time of exposure of the cells to the reagent and to the amount of deposition per cell than to the number of cells containing red formazan. No differential inhibition of acetylene reduction versus CO₂ fixation was observed. Autoradiography showed that CO₂ fixation occurred only in vegetative cells. Heterocysts caused a darkening of nuclear emulsions (chemography). This observation has been employed by others as an index of reducing activity in these cells. DCMU inhibited the acetylene reducing capacity of alga isolated from dark pretreated fronds more rapidly and to a greater extent than that in alga isolated from light pretreated fronds. Ammonia in excess of 5 mM was required before any inhibition of acetylene reduction was observed under either aerobic or anaerobic conditions in the light.     

Enzymes of Purine Biosynthesis and Catabolism in Glycine max: I. COMPARISON OF ACTIVITIES WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING NODULE DEVELOPMENT

During the period examined from 12 to 63 days after planting, the ureides, allantoin and allantoic acid, were the predominant nitrogenous solutes in the xylem exudate of soybeans (Glycine max [L.]) growing solely on symbiotically fixed nitrogen, accounting for approximately 60% and greater than 95% of the total nitrogen in the xylem exudate before and after the onset of active nitrogen fixation, respectively. For plants between 18 and 49 days of age, the apparent rate of ureide export estimated from concentrations of ureides in xylem exudate collected over a period of one hour was closely related to the rate of nitrogen fixation estimated from measurements of C₂H₂ reduction by nodulated root systems. After this time, the apparent rate of ureide export per plant continued to increase, reaching a maximum value at day 63 of 12 micromoles per plant per hour, even though the rate of C₂H₂ reduction per plant declined approximately four-fold. The most probable pathway for the biosynthesis of ureides involves the catabolism of purines. The levels of phosphoribosylpyrophosphate (PRPP) synthetase, which catalyzes the formation of the PRPP required for purine synthesis, increased in parallel with the rates of nitrogen fixation (C₂H₂) from day 18 reaching a maximum value of 13.9 micromoles per plant per hour at day 49, and then both activities declined rapidly. During the period of active nitrogen fixation the ratio of PRPP synthesis estimated from measurements of PRPP synthetase activity in cell-free extracts to the apparent rate of ureide export was between 1 and 2. The activities of the enzymes of purine catabolism, xanthine dehydrogenase, uricase, and allantoinase, increased in parallel with the increases in nodule mass and the export of ureides with maximum activities of 13, 119, and 79 micromoles per plant per hour, corresponding with apparent rates of ureide export in the range of 9.5 to 11.9 micromoles per plant per hour. These results demonstrate that there is a close association between nitrogen fixation, PRPP synthetase activity, and ureide export in soybeans and support the proposal that recently-fixed nitrogen is utilized in the de novo synthesis of purines which are subsequently catabolized to produce the ureides.     

The Azolla, Anabaena azollae Relationship: II. Localization of Nitrogenase Activity as Assayed by Acetylene Reduction

Anaerobic (microaerophilic) acetylene reduction by Azolla caroliniana Willd. was dependent on light and saturated at approximately 450 foot candles. Maximum rates of acetylene reduction were 60 nmoles/mg chlorophyll minute. However, rates of 25 to 30 nmoles/mg chlorophyll minute were more common.     

Problems of the Acetylene Reduction Technique Applied to Water-Saturated Paddy Soils

The acetylene reduction assay for the measurement of N₂ fixation in a water-saturated paddy soil is limited by the slow diffusion of acetylene and ethylene. In laboratory incubation tests, vigorous shaking after the assay period is needed to release ethylene into the gas within the assay vials. Shaking prior to the incubation is also effective for dissolving acetylene in the water-saturated soil. However, a water-saturated soil depth of less than 10 mm during incubation is recommended. In field assays, some amounts of ethylene remain in the water-saturated soil phase of the acetylene reduction assay chamber, but stirring the water-saturated soil before sampling reduces the amount of ethylene remaining in soil. Evidence of a downward movement of acetylene and an upward movement of ethylene through rice plants was obtained. Because of the rapid transfer of acetylene to rice plant roots, an in situ acetylene reduction assay covering a rice hill is likely to detect nitrogen fixation in the proximity of roots where acetylene is easily accessible. Acetylene introduction to the water-saturated soil phase prior to assay did not greatly increase the acetylene reduction rate. Carbon dioxide enrichment in the assay chamber did not enhance nitrogen fixation in a paddy including rice and algae during a 1-day cycle.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.