Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Heterotrophic nitrogen fixation (acetylene reduction) associated to flooded rice: A modified measurement technique in the field

A modifiedin situ technique for measuring heterotrophic nitrogen fixing (acetylene reducing) activity associated to rice is proposed. Ethylene evolution rates measured in opaque cylinders covering the stems of rice plants which have been cut 10 cm over the water level were found independent of the diurnal cycle. Cutting of the leaves resulted in decreased variation between plants and suppression of the acceleration of ethylene evolution rate after 12 h incubation as compared to intact plants. In both systems ethylene evolved was swept by a current of methane and the molar ratio between methane and ethylene was stabilized after 12 h. Methane evolution rates remained stable during 12 h and more than 24 h in whole plants and cut plants respectively. It is suggested that alteration in the active gas transport system after 12 h incubation under 10% acetylene may lead to erroneous evaluation of the actual ethylene production in the root's environment. The average values of ethylene evolution rates by cut plants between 12 and 24 h of incubation may be used for comparative studies of nitrogen fixing activity associated to flooded rice.     

pH、温度、NH4+和盐度对圆果雀稗内生固氮菌固氮特性的影响

采用乙炔还原法对从圆果雀稗(Paspalum orbiculare)根、茎中分离到的5株内生固氮菌(POJy11、POG12、POGy22、POGy612、POG4)的固氮特性进行了研究。结果表明,POGy612、POG4固氮的pH适应范围是6.0-10.0,其余3株菌为6.0-8.0;POGy22固氮的最适温度为35℃,其余4株菌为30℃;随着NH4 浓度和NaCl浓度的升高,菌株固氮活性总体呈下降趋势,其中POGy22的固氮活性受NH4 抑制最明显,NH4 浓度为1.0 mmol/L时,已无固氮活性,但POGy612在NH4 浓度为2.5 mmol/L时的固氮活性最高,达到1 045.0 nmol ml-1h-1,POJy11、POGy22在NaCl浓度为0.5%时的固氮活性最高,分别达到735.5 nmol ml-1h-1、703.0 nmol ml-1h-1。     

Nitrogen-fixing root nodules inDatisca Cannabina L.

Summary The occurrence of root nodules inDatisca cannabina is reported. The nodules are typically of the Alnus type, forming dichotomously-branched coralloid clusters. The enlarged cortical cells contain vesicle clusters of the endophyte. The nodules reduced acetylene to ethylene at a rate of 5.5 moles ethylene per g fresh wt of nodules per hour.     

Non-destructive semi-quantitative determination of acetylene reduction in the field

Summary Direct injection of acetylene into soil around plant roots, followed by determination of ethylene/acetylene ratios in the soil atmosphere has been tested as a rapid, non-destructive method of estimating acetylene reducing activity. In pots of artificial media as well as in field soil, the ratios determined within 10 min. after injection were significantly correlated with the rates of acetylenedependent ethylene production in detached roots. The method may be useful in preliminary screening of large numbers of plant-bacteria combinations.     

Effect of lime additions to acid strip-mine spoil on survival,growth and nitrogen fixation (acetylene reduction) of several woody legume and actinomycete-nodulated species

Summary An acid mine spoil in Southern Indiana was amended with lime (CaCO₃) (0.0, 12.5, 25 and 39t/ha) and planted withElaegnus umbellata Thunb.,Alnus glutinosa Gaertn.,Robinia pseudoacacia L.,Robinia fertilis Ashe, Arnot,Myrica pensylvania Lois,Caragana arborescens L. andShepherdia argentea Nutt. Survival and soil data were collected periodically and plants were harvested 15 months after planting. Nodule and top dry weights were determined and acetylene reduction assays performed on the nodules.Addition of lime caused significant increases in pH, and 39 t/ha of lime were required to maintain a pH above 5.5. Survival of plant material was greatest at the highest lime addition, although response of individual species varied.Elaeagnus umbellata, R. pseudoacacia, R. fertilis Arnot, andA. glutinosa appeared more tolerant of the harsh conditions. OnlyC. arborescens showed a linear increase in top dry weight due to lime addition.Alnus glutinosa andS. argentea achieved statistically the same growth regardless of pH, andR. fertilis Arnot andE. umbellata did not increase in top dry weight above an addition of 25 t/ha.Robinia pseudoacacia achieved maximum top dry weight at 25 t/ha, whereasM. pensylvanica growth declined with increasing pH. Nodule dry weights increased with increasing pH; however,S. argentea showed greater nodule dry weights at lower lime levels. Acetylene reduction rates increased with lime addition.Elaegnus umbellata did not respond above 25 t/ha lime, whereasA. glutinosa did not show an increase until this point.     

Acetylene reduction,H2 evolution and 15N2 fixation in the Alnus incana-Frankia symbiosis

Acetylene reduction, ¹⁵N₂ reduction and H₂ evolution were measured in root systems of intact plants of grey alder (Alnus incana (L.) Moench) in symbiosis with Frankia. The ratios of C₂H₂: ¹⁵N₂ were compared with C₂H₂:N₂ ratios calculated from C₂H₂ reduction and H₂ evolution, and with C₂H₂:N₂ ratios calculated from accumulated C₂H₄ production and nitrogen content. It was possible to calculate C₂H₂:N₂ ratios from C₂H₂ reduction and H₂ evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched ¹⁵N could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H₂ evolution in combination with C₂H₂ reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.     

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

Summary An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.     

Relationship between porewater organic carbon content, sulphate reduction and nitrogen fixation (acetylene reduction) in the rhizosphere of Zostera noltii

Depth profiles of nitrogen fixation (acetylene reduction), sulphate reduction, NH ₄ ⁺ concentration and porewater volatile fatty acids concentrations were measured in Zostera noltii colonised sediments in the Bassin d'Arcachon, France in March 1994. Acetylene reduction activity (ARA) was detectable throughout sediment profiles. Addition of sodium molybdate (20 mmol l^–1) a specific inhibitor of sulphate reduction to slurries inhibited ARA by >75% inferring that sulphate-reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. The peak of ARA was coincident with that of sulphate reduction and a relatively constant relationship of 40 mole sulphate reduced per mole acetylene reduced was recorded throughout the profiles. From this ratio it was calculated that at least 17% of the ATP yield from sulphate reduction would be required to support the measured rates of nitrogen fixation (acetylene reduction).Acetate was the dominant constituent of the porewater volatile fatty acids pool, accounting for >90% of the total pool as measured by HPLC. Concentrations of porewater acetate recorded by HPLC were compared with those measured using an enzymatic technique and these data indicate that approximately 10% of the total porewater acetate pool was not available to microbial metabolism. Profiles of porewater acetate concentrations measured by both techniques were similar to those recorded for both ARA and sulphate reduction and thus acetate oxidation may fuel these activities.     

Periphyton nitrogenase activity as an indicator of wetland eutrophication: spatial patterns and response to phosphorus dosing in a northern Everglades ecosystem

The use of periphyton nitrogenase activity (biological N₂ fixation) as an indicator of wetland P impact was assessed using patterns of nutrient content (C, N, P, Ca, Mg, K, Fe, and Mn) and acetylene reduction (AR) in floating cyanobacterial periphyton mat (metaphyton) communities of a P-enriched portion of the Florida Everglades, USA (Water Conservation Area-2A, WCA-2A). Spatial patterns of nutrients indicate the enrichment of floating mat periphyton N, P, Fe, and K, and the reduction of Mn and TN:TP in enriched marsh areas. In highly enriched areas, floating mat periphyton AR was approximately threefold greater than that in less enriched, interior marsh zones. Multiple regression models indicated AR dependence on P in eutrophic WCA-2A areas while the AR of more interior marsh periphyton mats was more closely related to tissue levels of Ca and Fe. Nitrogenase activity of floating mat periphyton from P-loaded mesocosms revealed a significant enhancement of N₂ fixation in samples receiving approximately 2–3 mg P m⁻² of cumulative P dosing or with biomass TP content of 100–300 mg kg⁻¹. At P contents above the optimum, mat periphyton AR was suppressed possibly as a result of changes in species composition or increased levels of NH₄⁺. After 3 years of dosing, consistently high AR occurred only at low rates of P enrichment (0.4–0.8 g P m⁻² yr⁻¹), and the patterns appeared to be seasonal. These findings agree with the hypothesis that P availability is a key determinant of nitrogenase activity in aquatic systems, and thus, may support the use of periphyton nitrogenase to indicate P impacts in P-limited systems. These results also demonstrate the potential existence of a P threshhold for biogeochemical alteration of periphyton mat function in the Everglades, and that cumulative loading of limiting nutrients (i.e., P), rather than instantaneous concentrations, should be considered when evaluating nutrient criteria.     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Nitrogen-fixing symbiosis between photosynthetic bacteria and legumes

Rhizobia having photosynthetic systems form nitrogen-fixing nodules on the stem and/or root of some species of the legumes Aeschynomene and Lotononis. This review is focused on the recent knowledge about the physiology, genetics and role of the photosystem in these bacteria. Photosynthetic electron transport seems to involve reaction centers, soluble cytochrome c2 and cytochrome bc1. Anaerobically, the electron transport system becomes over-reduced. The photosynthesis genes have been partially characterized; their organization is classical but their regulation is unusual as it is activated by far-red light via a bacteriophytochrome. This original mechanism of regulation seems well adapted to promote photosynthesis during stem symbiosis. Photosynthesis plays a major role in the efficiency of stem nodulation. It is also observed that infrared light stimulates nitrogen fixation in nodules containing photosynthetic bacteroids, suggesting that photosynthesis may additionally provides energy for nitrogen fixation, allowing for more efficient plant growth. Other aspects of these bacteria are discussed, in particular their taxonomic position and nodulation ability, the role of carotenoids and the potential for application of photosynthetic rhizobia in rice culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selenate reduction by a Pseudomonas species: a new mode of anaerobic respiration

The high levels of selenium (selenate, selenite) in agricultural drainage water in the San Joaquin Valley of California, which have led to environmental problems, might be lowered if the selenate/selenite could be reduced to elemental insoluble selenium. Two organisms have been newly isolated which do this in anaerobic coculture. One, a strictly anaerobic, Gram-positive rod, reduces selenite to elemental selenium. The other, a Pseudomonas species, was shown to respire selenate to selenite. Cells grown anaerobically in Minimal Medium on acetate plus selenate oxidized 14C-acetate to 14CO2 with concomitant reduction of selenate to selenite and small amounts of elemental selenium.     

Growth and nitrogen fixation ofAeschynomene under water stressed conditions

Summary Studies on the tolerance ofAeschynomene americana L. to periods of flooding or soil moisture deficit were conducted in an attempt to elucidate nitrogen fixation as affected by soil moisture. Nitrogenase activity was not reduced significantly in pot-grown Aeschynomene plants subjected to flooding in greenhouse conditions. After 20 days of withholding water from the soil, nitrogenase activities of the drought-stressed plants were much lower than those of either the well-watered or flooded plants. Leaf water potentials were similar in flooded and control plants; however, the droughted plants had leaf water potentials that were 4 bars lower than those of the control plants. Aeschynomene plants were tolerant to long-term periods of flooding, but exhibited a reduction in nitrogenase activity and leaf water status when subjected to soil moisture deficits.     

Delayed inoculation and starter nitrogen for enhancing early growth and nitrogen status ofLespedeza cuneata

Summary Two growth chamber experiments were conducted to determine the response ofLespedeza cuneata (Dumont) G. Don. (sericea lespedeza) to delayed inoculation and low levels of nitrogen fertilization. Nitrogen was supplied either as NH ₄ ⁺ or as NO ₃ ^– in solution. At 0.5 and 5.0 ppm nitrogen early growth and N₂(C₂H₂) fixation was inhibited by NH ₄ ⁺ and promoted by NO ₃ ^– . Inoculation at seeding did not negatively affect growth prior to the onset of N₂(C₂H₂) fixation. Delayed inoculation until the trifoliate stage thus did not increase growth or N₂ fixation during the first 40 days of growth. After 40 days, specific nitrogenase activity was highest for plants inoculated at the first trifoliate stage of growth. In contrast, growth and total shoot nitrogen accumulation were higher in plants inoculated at planting. The experimental results suggest that delaying inoculation is not a useful technique for improving early growth ofL. cuneata for surface mine reclamation.     

Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed cultures

Discharge of nitrate and ammonia rich wastewaters into the natural waters encourage eutrophication, and contribute to aquatic toxicity. Anaerobic ammonium oxidation process (ANAMMOX) is a novel biological nitrogen removal alternative to nitrification-denitrification, that removes ammonia using nitrite as the electron acceptor. The feasibility of enriching the ANAMMOX bacteria from the anaerobic digester sludge of a biomethanation plant treating vegetable waste and aerobic sludge from an activated sludge process treating domestic sewage is reported in this paper. ANAMMOX bacterial activity was monitored and established in terms of nitrogen transformations to ammonia, nitrite and nitrate along with formation of hydrazine and hydroxylamine.     

Effects of available P and N:P ratios on non-symbiotic dinitrogen fixation in tallgrass prairie soils

Summary Prescribed burning is a major control over element cycles in Tallgrass prairie (Eastern Kansas, USA). In this paper we report potential effects of fire on nonsymbiotic nitrogen fixation. Fire resulted in additions of available P in ash, which may stimulate nitrogen fixation by terrestrial cyanobacteria. Cyanobacterial nitrogenase activity and biomass responded positively to additions of ash or P in laboratory assays using soil. Further assays in soil showed that cyanobacteria responded to changes in available N:available P ratio (aN:P) across a range of concentrations. Nitrogen fixation rate could be related empirically to aN:P via a log-linear relationship. Extrapolation of laboratory results to the field yielded a maximal estimate of 21 kg N ha^-1 y^-1. Results support arguments from the marine and terrestrial literature that P availability is central to regulation of ecosystem N budgets.     

15N2 incorporation and acetylene reduction by Azospirillum isolated from rice roots and soils

Summary Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by¹⁵N₂ incorporation and C₂H₂ reduction. C₂H₂ reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in¹⁵N₂ incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N₂-fixing ability and that plant genotype is of importance for optimal associations.     

Nitrogen-fixing azotobacters from mangrove habitat and their utility as marine biofertilizers

A dearth of information is available for nitrogen-fixing bacteria in coastal mangroves, and hence, the present study has been undertaken to analyse 44 root and associated soil samples, derived from a mangrove habitat of southeast coast of India. The root samples exhibit high counts of total heterotrophic bacteria and azotobacters along with high rates of nitrogen fixation, as compared to the rhizosphere soil samples. Among the plant species, Bruguiera cylindrica records high microbial counts and nitrogen fixation. From the samples analysed, three species of Azotobacter, viz., A. chroococcum, A. virelandii and A. beijerinckii were isolated, purified and identified. These species exhibit high growth, nitrogen fixation and in vitro production of phytohormone (Indole Acetic Acid, IAA) at NaCl salinity of 30 g l⁻¹. The azotobacters, which were inoculated with Rhizophora seedlings, increased significantly the average root biomass up to by 98.2%, the root length by 48.45%, the leaf area by 277.86%, the shoot biomass by 29.49% as compared to controls and they also increased the levels of total chlorophylls and carotenoids up to by 151.0% and 158.73%, respectively. Thus, azotobacterisation is beneficial in raising vigorous seedlings of mangroves in coastal wetlands.