Acetylene Reduction Rates Research Articles

Studies on two classes of rifampicin-resistant mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Two types of nitrosoguanidine-induced rifampicin-resistant mutants ofNostoc muscorum were isolated and characterized. Compared with the wild type, the strainrif-1 (rifrhet+nif+blu) revealed high growth rate, heterocyst frequency (10%–12%), nitrogenase activity, phycocyanin pigment, photosynthetic O2 evolution, and higher activities of phosphoribulokinase and Fd-NADP+-oxidoreductase. The heterocyst spacing pattern in the mutant was altered and did not respond to 7-azatryptophan, β-2-thienylalanine, rifampicin, andl-methionine-dl-sulfoximine treatment. The second type of mutantrifr-2 (rifrhet+nif−) did not show nitrogenase activity and was unable to grow on molecular nitrogen even under microaerobic conditions, although it produced heterocysts (6%–7%) under these conditions of incubation. The pattern of macromolecular synthesis, particularly of RNA and protein, the rate of acetylene reduction, and photosynthetic O2 evolution were not affected in the mutant strains with the treatment of drug. The characteristics of the mutants reflected the possibility of pleiotropic mutation and also suggested that therif marker is most likely associated in close genetic proximity with regulatory gene(s) ofhet andnif system.

Variation in nitrogen fixation among populations ofFrankia sp. andCeanothus sp. in actinorhizal association

Wildland shrub improvement is needed for sound range and disturbed land revegetation practice. The possibility of selecting superior N2-fixingFrankia-Ceanothus spp. actinorhizal associations was examined. Greenhouse tests were used to expose various soil-borne microsymbiont andCeanothus sp. population accessions in reciprocal combination. The acetylene reduction rate was used as a measure of N2-fixation capacity. There was no significant interaction between host and microsymbiont regardless of source for all variables measured. The acetylene reduction rate, nodule number and mass, plant biomass, and root: shoot ratio were significantly different among soil sources. The acetylene reduction rate was not significantly different amongCeanothus sp. accessions. Neither was it strongly correlated with other variables. It was concluded that the N2-fixation rate is more a function ofFrankia sp. than the hostCeanothus sp. in actinorhizal associations. It appears possible to select soil sources with superior N2-fixing microsymbiont populations.

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO(2) fixation and in vivo NH(4) assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO(2) fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.

Asymbiotic dinitrogen fixation by tundra in the Imnavait Creek drainage, Alaska, USA

N2 fixation by free-living microorganisms was investigated at an intensively studied low Arctic site near Toolik Lake in the northern foothills of the Brooks Range, Alaska, during July 1987. Four characteristic vegetation associations along an elevational gradient were assayed using minimally disruptive in situ acetylene reduction assay methods. The acetylene reduction rates did not differ significantly among vegetation associations. The mean rate for the site was 9.60 μmol m−2 h−1 or 90 μg N m−2 day−1, which is within the range of values given for other Arctic and alpine tundra studies. The complex microtopography and resulting patchy distribution of free-living and phycobiont diazotrophs is the most likely cause of the high spatial variability in acetylene reduction activity. Rates were most variable among samples from the lowest position, a riparian site. The potential contribution of heterotrophic diazotrophs was examined through a laboratory enrichment study. Soils from the two lower slope positions showed dramatic responses to added C, suggesting that heterotrophs may contribute fixed N2 to this system.

A survey of nitrogen fixation (acetylene reduction) associated with Typha in Minnesota

The acetylene-reduction technique was used on soil–root cores to examine the three species of Typha (Typhaceae) found in Minnesota for differences in rates of nitrogen fixation by associated rhizosphere diazotrophs. In 20 cattail stands sampled in 1985, Typha angustifolia showed a tendency for lower acetylene-reduction activity per ramet, with an average of 2.9 μmol∙ramer−1∙d−1, than T. latifolia and T. ×glauca, with averages of 6.5 and 7.1 μmol∙ramet−1∙d−', respectively. Regression analysis showed no significant differences in these values due to the variability within as well as between stands, and fixation rates were uncorrelated with the pH, soil percent carbon, and soil moisture of the study stands. In 1986, in situ assays of soil–root cores of ramets of T. angustifolia and T. latifolia from adjacent monospecific stands produced average acetylene-reduction rates of 1.4 for T. angustifolia and 1.8 μmol∙ramet−1∙d−1 for T. latifolia, with a substantial reduction in variability within stands over the 1985 data. It is estimated that, contrary to previously published reports, populations of Typha may receive as little as 1–2% of their annual nitrogen requirement from nitrogen fixation.

Effects of organic substrates and chloramphenicol or nalidixic acid on acetylene reduction associated with roots of intact maize and sorghum plants

To study the role or organic substrate availability as a factor limiting associative N2-fixation we measured acetylene reduction (AR) associated with roots of intact maize and sorghum plants before and after adding organic substrates to the nutrient solution in a hydroponic system. Chloramphenicol (Cam) or nalidixic acid (NA) was added along with the substrate to determine whether bacterial protein synthesis or cell replication was necessary to support increased AR following amendment. The grasses were grown in pots in a greenhouse or on a light bench for 4–6 weeks, and then brought into the laboratory to measure AR. Intact plants were separated from soil and transferred into plastic cylinders containing an N-free nutrient solution. The roots were isolated from the shoots by a silicone rubber seal and exposed to oxygen concentrations of 0–10 kPa. Rates of AR were measured before and after adding 0.01–0.10% (w/v) carbon as glucose, malate, succinate, ethanol, acetate, glutarate, propionate, or resorcinol. Only resorcinol and ethanol failed to substantially increase AR activity. Rates of AR increased by 1.5-to 2-fold within 2h and by 5-to 15-fold after 24h. Cam and NA prevented the stimulation of AR by glucose, but neither inhibitor caused AR associated with unamended plants to decrease. We conclude that the highly variable rates of AR that have been reported for associative symbioses, even under well-controlled conditions were governed to a large extent by the amount and type of organic substrates exuded by the roots. Proliferation of diazotrophs appeared to be necessary to increase root-associated AR activity but not to maintain a constant level of activity.

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.

Dinitrogen Fixation in the Wet Meadows and Emergent Zones of Two Nebraska Sandhills Lakes

-Dinitrogen fixation (acetylene reduction) activity was determined along a moisture gradient transect of a neutral-slightly alkaline lake (Island Lake) and an alkaline lake (Goose Lake) in western Nebraska during the summers of 1981 and 1982. Vegetation graded from Carex meadow to Typha to Scirpus emergent zones along each of the 50-m transects. Abundant growth of cyanobacteria occurred on exposed soil surfaces and epiphytically below the water line in the emergent zones. The Carex meadow exhibited relatively low and variable rates of acetylene reduction, whereas the Typha and Scirpus zones had consistently high rates of activity. Estimated mean nitrogen influx rates (kg ha-' yr-1) for the 1981 and 1982 seasons were consistently higher at Goose Lake (Carex, 0.5; Typha, 10.143.8; Scirpus 8.5-13.5) compared to Island Lake (Carex, 0.1; Typha, 0.9-10.6; Scirpus, 2.95.7). The higher rates of activity at the Goose Lake site are most likely due to the alkaline pH which is considered to favor cyanobacteria. The expansive wet meadow-emergent zones bordering the typically shallow Sandhills lakes support microbial populations which contribute large amounts of biologically fixed nitrogen to these aquatic ecosystems.

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids.

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.

Impact of soil environmental factors on rates of N2-fixation associated with roots of intact maize and sorghum plants

We have evaluated the effects of oxygen partial pressure (pO2), combined nitrogen, and the availability of organic substrates on nitrogen fixation (acetylene reduction) by bacteria associated with the roots of intact maize and sorghum plants. We also investigated the possibility of enhancing associative nitrogen-fixation by inoculating the soil in which the plants were grown with Azospirillum. Acetylene reduction (AR) activity was greatest when roots of intact plants were exposed to pO2 between 1.3 and 2.1 kPa. Field-grown and greenhouse-grown plants supported similar levels of activity. Respiration inhibitors (2,4-dinitrophenol and sodium azide) eliminated AR activity at 2kPa O2, whereas a fermentation inhibitor (sodium fluoride) only partially reduced the activity. Acetylene reduction activity was rapidly (1–3 h) inhibited by NH4 +, NO3 -, and NO2 - at concentrations of 4–20mg N1-1. Rates of AR varied substantially among individual plants in each experiment and between experiments. Amendment with any of several organic substrates greatly increased AR activity when rates were low, suggesting that the lack of activity was caused by a shortage of available carbon in the rhizosphere. Inoculation with Azospirillum failed to increase rates of AR associated with maize plants. In several experiments the indigenous bacteria associated with uninoculated plants exhibited greater activity than the bacteria associated with inoculated plants.

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N2 fixation and a number of hypotheses concerned with NO3− assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea (Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N‐free medium, followed by 1 week in medium supplemented with 0, 5 or 10 mM KNO3 before harvesting. NO3 was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO3 but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO3 and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO3. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO3 inhibition of N2 fixation since NO3 affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO3 inhibition of N2 fixation suggests that NO3 assimilation may be only indirectly involved in the inhibition phenomenon.

Soybean nodule gas permeability, nitrogen fixation and dirunal cycles in soil temperature

While diurnal cycles in nitrogen fixation rates are sometimes assumed to result from diurnal variation in photosynthetically active radiation, contradicting evidence exists that indicate soil temperature is the primary environmental influence. These studies assessed the significance of temperature on soybean nitrogen fixation under field conditions. Two groups of intact field-grown soybean plants, one at ambient and the other exposed to a 10°C diurnal variation in soil temperature, were nondestructively assayed for acetylene reduction rates. Activity was closely associated with soil temperature (R2=0.85), even when temperature was 12 h out of phase with ambient. Data were also obtained to determine if the effects of rhizosphere temperature on nitrogen fixation are mediated through an effect on the nodule oxygen permeability. Nodule oxygen permeability of intact, aeroponically grown soybean was closely correlated with the diurnal changes in temperature (R2=0.90).

Acetylene reduction in black alder seedlings as affected by direct and indirect moisture deficits using a split-pot growing system

Black alder seedlings inoculated with one of two strains of nodule endophyte Frankia were used to evaluate the effects of direct soil moisture stress using a split-pot growing system. Seedlings were either watered in both rooting zones (control); watered only in the lower rooting zone (only nodulated root tissue exposed to dry soil); or no water to either rooting zone. The split-pot growing system was successful in exposing nodulated roots of black alder seedlings to dry environments (direct soil moisture stress) while keeping the host plant fully hydrated. There were no significant differences between strains of Frankia in their response to direct soil moisture stress. Transpiration rates and leaf water potentials were similar for well watered controls and seedlings watered only from below. Transpiration rates for seedlings receiving no water were significantly lower (24%) than those of the controls; seedlings watered only from below had a non-significant 3.9% reduction. Despite full shoot hydration, photosynthesis was reduced 56% from control levels when seedlings were watered from below. There were no significant differences for leaf conductance between treatments. Direct soil moisture stress had no significant effect on acetylene reduction. However, acetylene reduction rates for fully stressed seedlings were 44% lower than those of the controls. Direct soil moisture stress or complete plant stress had no significant effect on nodule sugar content.

Response tobradyrhizobiumstrain of peanut cultivars grown under iron stress

Abstract An Fe efficient (Arachis hypogaea L. cv. 71–234) and an Fe inefficient (cv. Tainan 9) peanut cultivar were subjected to four inoculation treatments (NH4NO3 without inoculation, NC 92, TAL 1000 and a mixture of these two Bradyrhizobium sp. (Arachis) strains with THA 205) and grown under Fe stress conditions in complete nutrient solution including 0.2 mM NH4NO3 and 0.012 mM FeEDDHA at pH 8.6 maintained by addition of 5 mM NaHCO3 and solid CaCO3. During the first four weeks, cv. 71–234 grew better than cv. Tainan 9 in all inoculation treatments but by eight weeks these differences had largely disappeared. By contrast, inoculation treatments had no effects on shoot and root dry matter at four weeks but large effects at eight weeks when plants inoculated with NC 92 alone or in a mixture had similar or more dry matter than those given NH4NO3 and double that of those with TAL 1000 alone. At four weeks, both cultivars had 250, 150, and 3 nodules per plant and acetylene reduction rates of 27, 17, <1 nmol/...

Altitudinal variation in nitrogenase activity of the Himalayan alder naturally regenerating on landslide‐affected sites

SUMMARYNatural regeneration, root nodule biomass and nitrogenase activity (acetylene reduction) of Alnus nepalensis D. Don (alder) were estimated at ten different landslide‐affected elevated (1000–2500 m) sites in the Kalimpong forest division of the Eastern Himalayas. Alder regeneration was best in the middle zone (1500–2000 m) of this altitude range. Nitrogenase activities were between 5 and 19 μmol C2H4 g−1 nodule d. wt h−1 in a peak activity month (July). The activity was largely dependent on mean soil temperature and root nodule moisture. The highest rate of acetylene reduction was found in the rainy season (July) at the 1830 m elevation site. Nitrogenase activity was negligible at night and increased during the day with a midday maximum. Alder regeneration, seedling growth, root nodule biomass and nitrogenase activity were temperature dependent along the gradient of altitude and fell off below 1500 m and above 2000 m.

Time Course of Acetylene Reduction in Nodules of Five Actinorhizal Genera

The rate of acetylene reduction was measured as a function of time after addition of 10% acetylene in Alnus, Casuarina, Ceanothus, Datisca, and Myrica. The maximum rate occurred after 45 to 60 seconds and was maintained for an additional 0.5 to 4 minutes before a decline in rate to 30 to 90% of the maximum. The rate then recovered to a value of 63 to 98% of the maximum. Removal of the shoot and lower roots did not affect nodule activity.

Effect of potassium on nitrogen fixation, nitrogen transport, and nitrogen harvest index of bean

Abstract Four cultivars of Phaseolus vulgaris L., Carioca, Venezuela‐ 350, Rio Tibagi and Negro Argel, were grown to maturity in a glass house with a low (1 mol m‐3) and a high (4.5 mol m‐3) potassium supply. Potassium stimulated nitrogenase activity (acetylene reduction) and the accumulation of ureides in pod walls of Carioca and Negro Argel, cultivars which had the highest rates of acetylene reduction. In three of the four cultivars the higher level of potassium nutrition resulted in a greater partitioning of above ground N to the seeds. The proportion of xylem sap N in the form of the ureides, allantoln and allantoic acid (allantoate), was only slightly increased by the higher rate of potassium supply. Seed yields were increased by high potassium in the cultivars Venezuela‐350 and Negro Argel. The results suggest that potassium stimulates the transport of nitrogenous compounds to developing fruits.

Seed protein characterisation and nitrogen fixation rates in the chickpea mutant hyprosola and its parent

AbstractThe amino acid composition of the seed proteins of Hyprosola, a high protein, high yielding mutant of chickpea (Cicer arietinum L), differed little from that of its parental cultivar, Faridpur‐1. Oser's essential amino acid index was 76‐5 in the mutant and 76‐7 in the parent. Hyprosola seed has higher crude protein, extractable protein, globulin and albumin contents than Faridpur‐1. The mutant had an albumin/globulin ratio of 0‐23 compared with the parental value of 0‐21. The mutant oligomeric proteins differed quantitatively and qualitatively from the parent on cellulose acetate electrophoretograms. The polypeptide composition of globulins and albumins fractionated by SDS‐PAGE differed markedly in the intensity of many bands but not in the mobility of any polypeptides. Nitrogen fixation (acetylene reduction) rates were similar in the two cultivars, and in both hydrogen evolution utilised 45 % of the electron flow through nitrogenase.

Nitrogen Fixation on the Tropical Volcano, La Soufrière (Guadeloupe): Nitrogen Fixation, Photosynthesis and Respiration During the Prevailing Cloud/Shroud Climate By Stereocaulon Virgatum

AbstractStereocaulon virgatum Ach. has colonized lava flows deposited on the west flank of the volcano La Soufrière. The mean annual rate of acetylene reduction was 43·4 nmol C2H4 gdw−1 h−1 with maximum rates during the prevalent cloud/shroud meteorology of 101 and minimum rates during rare high insolation events of 0·63 nmol gdw−1 h−1. Percentage thallus moisture was the major variable controlling nitrogenase activity. During cloud/shroud conditions the upper 90% of the lichen canopy reduced 85% of the acetylene. Canopy shading reduced intra-canopy temperatures allowing the basal 10% of the canopy to fix nitrogen during insolation shocks. Basal portions of pseudopodetia exhibited reduced rates of ethylene production when exposed to canopy surface light intensities during cloud/shroud conditions. The recovery pattern of nitrogenase following desiccation during an insolation shock is presented. Rates of photosynthesis during cloud/shroud conditions were high, reaching 50% of those attained during saturating light intensities.

Rhizobiophage effects on Bradyrhizobium japonicum, nodulation and soybean growth

Rhizobiophage may potentially alter the ecological balance of competing rhizobial strains in soil. We determined the effect of a phage specific for Bradyrhizobium japonicum USDA 117 on the survival of two strains of B. japonicum (USDA 117 and 110) in soil. The phage was initially added to a sterile silt loam soil amended with the homologous (USDA 117) or the nonhomologus (USDA 110) rhizobia. The soil was assayed for the survival of both the phage and the rhizobia. The phage reduced the population of homologous rhizobia. This reduction was accompanied by a concurrent increase in phage titer. The phage had no significant effect on the soil population of the nonhomologous rhizobia. To examine the effects of the phage on nodulation and soybean [ Glycine max (L.) Merr.) growth, inoculated ( B. japonicum USDA 117) and noninoculated soybeans were grown in a sterile silt loam soil amended with two different concentrations of the phage. The phage significantly reduced nodule number, nodule weight and acetylene reduction. In soil inoculated with greater numbers of phage, the nodule number, nodule fresh weight and acetylene reduction were 10 nodules, 40 mg and 2340 nmol C 2 H 4 g −1 nodule h −1, respectively. The control plants inoculated with B. japonicum USDA 117, but without phage, had a nodule number of 44, a nodule weight of 144 mg, and an acetylene reduction rate of 7145 nmol C 2H 4 g −1 nodule h −1. To assay the selective effect of the phage on B. japonicum USDA 117, the phage was also inoculated into a nonsterile, phage- and rhizobia-free silt loam soil amended with B. japonicum USDA 117 and the nonhomologous B. japonicum USDA 110. The phage significantly reduced nodulation and N 2 fixation by soybeans inoculated with only B. japonicum USDA 117. No significant phage effect on these qualities was observed on plants inoculated with B. japonicum USDA 110 or a combination of B. japonicum USDA 110 and USDA 117. The phage also significantly decreased nodule occupancy by B. japonicum USDA 117 with a corresponding increase in occupancy by B. japonicum USDA 110.