Acetylene Reduction Assay Research Articles

Nitrogen Inhibits Nodulation and Reversibly Suppresses Nitrogen Fixation in Nodules of Alnus maritima

Symbiotic associations between Alnus maritima (Marsh.) Muhl. ex Nutt. (seaside alder) and actinomycetes in the genus Frankia Brunchorst result in root nodules in which atmospheric nitrogen (N) is fixed. The economic and environmental benefits of N fixation have led to interest in inducing root nodules during production of A. maritima. Because woody plants produced in nurseries typically are provided N fertilizer, our objectives were to determine how applied N influences nodulation of A. maritima and to characterize how short-term changes in root-zone N affect the function of nodules. Potted seedlings were grown in perlite that was inoculated with 30 mL of soil from the root zones of mature plants in their native habitat on the Delmarva Peninsula. Each pot was drenched once daily for 10 weeks with nutrient solution that contained ammonium nitrate at 10 concentrations from 0 to 8 mm. Plants that received no ammonium nitrate formed the most nodules, and nodulation decreased linearly as ammonium nitrate increased from 0.25 to 4 mm. Plants treated with ammonium nitrate at 4 or 8 mm formed nearly no nodules, while ammonium nitrate at 0.5 mm resulted in vigorous plants with an average nodule count of 70. In a second experiment, a population of nodulated seaside alders was established by irrigating seedlings in inoculated perlite once daily with 0.5-mm ammonium nitrate for 6 weeks. Plants then were provided ammonium nitrate at 0.5, 2, or 4 mm for 2 weeks. Acetylene-reduction assays showed suppressed nodule activity among plants provided 2- and 4-mm ammonium nitrate. Daily irrigation of those plants with N-free solution subsequently led to a rapid depletion of root-zone N and to a concomitant resurgence of nodule activity. These results demonstrate that N fertilization can be managed to promote nodulation of A. maritima and show that decreased nodule activity caused by short-term increases in root-zone N is reversible.

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO2 production) and nitrogenase activity (H2 production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N2:O2, thus under N2-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N2 deprivation that occur in acetylene reduction assays and during extended Ar:O2 exposures in H2 assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O2 from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO2/H2. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO2 produced per 2e− transferred by nitrogenase to reduction of N2. The results ranged from 2.6 to 3.4 mol CO2 produced per 2e−. Carbon cost expressed as g C produced per g N reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N2-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.

Growth, nitrogen fixation and mineral acquisition of Alnus sieboldiana after inoculation of Frankia together with Gigaspora margarita and Pseudomonas putida

The role of tetrapartite associations among Frankia, Gigaspora margarita (an arbuscular mycorrhizal fungus), Pseudomonas putida (rhizobacterium), and Alnus sieboldiana in growth, nitrogen fixation, and mineral acquisition of A. sieboldiana was investigated. Seedlings of A. sieboldiana were inoculated with Frankia isolated from root nodules of alder, followed by inoculation of G. margarita and P. putida, and were grown for 5 months in a greenhouse. The seedlings inoculated with Frankia and G. margarita together produced the highest biomass of shoots and root nodules. Nitrogen-fixation activity, measured by acetylene reduction assay, was observed when Frankia was inoculated. The activity, on a per-nodule gram basis, decreased after G. margarita inoculation, but on a per-plant basis there was no significant difference in the activity among inoculation treatments. The mineral content in the seedlings changed after inoculation with Frankia, but not after inoculation with P. putida and/or G. margarita. The results showed a synergistic interaction among Frankia, the mycorrhizal fungus, and the rhizobacterium on the growth of A. sieboldiana.

The moderately halophilic bacterium Halomonas maura is a free-living diazotroph

Halomonas maura is a moderately halophilic bacterium which lives in saline soils and synthesises an exopolysaccharide known as mauran. Strain S-31 T grew in a nitrogen-free medium under an N 2 atmosphere; the acetylene reduction assay proved positive under specific conditions. We identified the nifH gene in this strain by using degenerate oligonucleotides designed from highly preserved gene sequences obtained from the alignment of a large number of nifH sequences from different microorganisms. Our results lead us to conclude that H. maura is capable of fixing nitrogen under microaerobic conditions.

Impacts of warming and fertilization on nitrogen-fixing microbial communities in the Canadian High Arctic

The impacts of simulated climate change (warming and fertilization treatments) on diazotroph community structure and activity were investigated at Alexandra Fiord, Ellesmere Island, Canada. Open Top Chambers, which increased growing season temperatures by 1–3 °C, were randomly placed in a dwarf-shrub and cushion-plant dominated mesic tundra site in 1995. In 2000 and 2001 20N:20P 2O 5:20K 2O fertilizer was applied at a rate of 5 g m −2 year −1. Estimates of nitrogen fixation rates were made in the field by acetylene reduction assays (ARA). Higher rates of N fixation were observed 19–35 days post-fertilization but were otherwise unaffected by treatments. However, moss cover was significantly positively associated with ARA rate. NifH gene variants were amplified from bulk soil DNA and analyzed by terminal restriction fragment length polymorphism analysis. Non-metric multidimensional scaling was used to ordinate treatment plots in nifH genotype space. NifH gene communities were more strongly structured by the warming treatment late in the growing season, suggesting that an annual succession in diazotroph community composition occurs.

NITROGEN FIXATION INCREASES WITH SUCCESSIONAL AGE IN BOREAL FORESTS

There is little understanding of successional dynamics of N fixation in northern boreal forests. Recent evidence suggests that N fixation by cyanobacteria in association with the common feather moss Pleurozium schreberi contributes to a significant proportion of the total N economy. The purpose of the work herein was to determine how time since last fire influences N fixation rates in boreal forests. We evaluated seasonal N fixation rates on a total of 12 natural forest preserves varying in time since last fire (35–355 years). Each site was monitored for N fixation activity using a calibrated acetylene reduction assay. Nitrogen fixation rates were found to increase linearly with time since fire. This increase in N fixation with succession is likely a function of degree of colonization by cyanobacteria and site factors such as presence of available N. Surface applications of 4.5 kg N·ha−1·yr−1 as NH4NO3 were found to eliminate N fixation while applications of P resulted in only a slight and temporary increase of N fixation rates. In contrast to common observation our findings suggest that N fixation in boreal forests becomes more important in late succession. Limited N availability in late succession is clearly one of the primary drivers of N fixation rates in boreal forest ecosystems. These findings may help to explain the origin of high rates of net N accumulation in soil unaccounted for at northern boreal sites.

Novel Endophytic Nitrogen-Fixing Clostridia from the Grass Miscanthus sinensis as Revealed by Terminal Restriction Fragment Length Polymorphism Analysis

Anaerobic nitrogen-fixing consortia consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K. Minamisawa, K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. Barraquio, N. Teaumroong, T. Sein, and T. Tadashi, Appl. Environ. Microbiol. 70:3096-3102, 2004). For this work, clostridial populations and their phylogenetic structures in a stand of the grass Miscanthus sinensis in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts. PCR primers and restriction enzymes were optimized for analyses of the plant clostridia. Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10(4) to 10(5) cells/g of fresh weight; they made up a large proportion of N2-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay. Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants. When phylogenetic populations from M. sinensis and the soil around the plants were compared, group II clostridia were found to exist exclusively in M. sinensis.

A viable microbial community in a subglacial volcanic crater lake, Iceland.

We describe a viable microbial community in a subglacial lake within the Grímsvötn volcanic caldera, Iceland. We used a hot water drill to penetrate the 300-m ice shelf and retrieved lake water and volcanic tephra sediments. We also acquired samples of borehole water before and after penetration to the lake, overlying glacial ice and snow, and water from a nearby subaerial geothermal lake for comparative analyses. Lake water is at the freezing point and fresh (total dissolved solids = 260 mg L(-1)). Detectable numbers of cells were found in samples of the lake water column and tephra sediments: 2 x 10(4) ml(-1) and 4 x 10(7) g(-1), respectively. Plate counts document abundant cold-adapted cultivable organisms in the lake water, but not in the borehole (before penetration) or glacial ice. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from genomic DNA extracted from Grímsvötn samples indicates that the lake community is distinct from the assemblages of organisms in borehole water (before penetration) and the overlying ice and snow. Sequencing of selected DGGE bands revealed that many sequences are highly similar to known psychrophilic organisms or cloned DNA from other cold environments. Significant uptake of 14C-labeled bicarbonate occurred in dark, low-temperature incubations of lake water samples, indicating the presence of autotrophs. Acetylene reduction assays under similar incubation conditions showed no significant nitrogen fixation potential by lake water samples. This may be a consequence of the inhibition of diazotrophy by nitrogen in the lake.

Can mushrooms fix atmospheric nitrogen?

It is generally reported that fungi like Pleurotus spp. can fix nitrogen (N2). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can fix N2. This was tested in vitro. Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean and P. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer from the fungus to the bradyrhizobial strain, because of high C cost of biological N2 fixation. This needs further investigations using 14C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N2. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N2 fixation. It is not the fungus that fixes N2 as reported earlier. Effective N2 fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications for future identification of as yet unidentified N2 systems occurring in the environment.

The Bunsen gas solubility coefficient of ethylene as a function of temperature and salinity and its importance for nitrogen fixation assays

The acetylene reduction assay is a common method for assessing nitrogen fixation in a variety of marine and freshwater systems. The method measures ethylene, the product of the conversion of the gas acetylene to its reduced form by nitrogenase. Knowledge of the solubility of ethylene in aqueous solution is crucial to the calculation of nitrogen fixation rates and depends on the temperature and salinity of the assay conditions. Despite the increasing interest in marine nitrogen fixation, no gas solubility (Bunsen) coefficients for ethylene in seawater are published to date. Here, we provide a set of equations and present semiempirically derived Bunsen coefficients for ethylene in water (ranging from 0.069 to 0.226) for a range of temperatures and salinities that are relevant for aquatic nitrogen fixation. We apply these data to nitrogen fixation scenarios at different temperatures and salinities and stress the importance of using accurate Bunsen coefficients in nitrogen fixation assays.

A Viable Microbial Community in a Subglacial Volcanic Crater Lake, Iceland

We describe a viable microbial community in a subglacial lake within the Grímsvötn volcanic caldera, Iceland. We used a hot water drill to penetrate the 300-m ice shelf and retrieved lake water and volcanic tephra sediments. We also acquired samples of borehole water before and after penetration to the lake, overlying glacial ice and snow, and water from a nearby subaerial geothermal lake for comparative analyses. Lake water is at the freezing point and fresh (total dissolved solids = 260 mg L–1). Detectable numbers of cells were found in samples of the lake water column and tephra sediments: 2 × 104 ml–1 and 4 × 107 g–1, respectively. Plate counts document abundant cold-adapted cultivable organisms in the lake water, but not in the borehole (before penetration) or glacial ice. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from genomic DNA extracted from Grímsvötn samples indicates that the lake community is distinct from the assemblages of organisms in borehole water (before penetration) and the overlying ice and snow. Sequencing of selected DGGE bands revealed that many sequences are highly similar to known psychrophilic organisms or cloned DNA from other cold environments. Significant uptake of 14C-labeled bicarbonate occurred in dark, low-temperature incubations of lake water samples, indicating the presence of autotrophs. Acetylene reduction assays under similar incubation conditions showed no significant nitrogen fixation potential by lake water samples. This may be a consequence of the inhibition of diazotrophy by nitrogen in the lake.

Protein content and dry weight of seeds from various pea genotypes

Pea seed protein content (SPC) and seed dry weight (SDW) are both influenced by genetic and environmental factors. To assess the variations of these within-plant traits between seeds, six genotypes were field tested. The sequential seed development at nodes along the main stem was determined. Nitrogen fixation was measured by the acetylene reduction assay (ARA). At maturity, protein content and dry weight were measured according to seed position on the plant. Individual protein content was determined by near-infrared transmission spectroscopy. The results show a significant difference in protein content between nodes of the genotypes Solara, L765 and L833. Protein content tended to decrease from the bottom to the top of the plant for these genotypes. The difference in protein content between the lowest and the uppermost node was about 26 g kg -1 for Solara, 40 g kg -1 for L765 and 49 g kg -1 for L833. There were also significant differences in dry weight between plant nodes for all genotypes, except Finale. In addition, the range of difference in dry weight between plant nodes was higher than that for protein content. Further, to determine the influence of morphological position on individual protein content and dry weight, multiple linear regression was established on node position, pod position on the node, and seed position within pods. The results showed that protein content and dry weight were not influenced either by within-pod seed position or pod position on the raceme. Moreover, protein content and dry weight were mainly influenced by node position on the main stem. However, for protein content, the effect of node position varied with genotype, indicating a genetic variability for this character. This genetic variability could be attributed to the difference between genotypes in the ability to maintain nitrogen fixation during the onset of seed filling. For dry weight, the decrease in seed weight for upper nodes of the plant also varied with genotype in relation to the duration of seed filling and the seed growth rate.

Long- and Short-term Effects of Nitrogen on Formation and Function of Root Nodules on Alnus maritima

Symbiotic associations between Alnus maritima (Marsh.) Muhl. ex Nutt. (seaside alder) and the actinomycete Frankia result in root nodules in which atmospheric nitrogen (N) is fixed. This has led to interest in producing seaside alders with minimal N fertilizer and in using the species on low-N soils. Our objectives were to determine how applied N influences nodulation and to characterize how short-term changes in root-zone N affect the function of established nodules. Seaside alders native to the Delmarva Peninsula (Alnus maritima subsp. maritima) were grown in perlite inoculated with soil from roots of indigenous plants. Plants were treated with N-free Hoagland solution supplemented with ammonium nitrate at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, and 8 mm for 10 weeks. Nodulation decreased as applied N increased. While plants treated with ammonium nitrate at 4 and 8 mm formed nearly no nodules, 0.5 mM resulted in vigorous, healthy plants that formed, on average, 70 nodules. In a second experiment, a nodulated population of seaside alders was established by treating seedlings with 0.5-mm ammonium nitrate in otherwise N-free Hoagland solution for 6 weeks. Plants then were provided ammonium nitrate at 0.5, 2, or 4 mm for two weeks. Acetylene-reduction assays showed that ammonium nitrate at 4 mm suppressed nodule activity. Daily irrigation with N-free solution subsequently led to a rapid depletion of root-zone N and a concomitant resurgence of nodule activity among plants previously provided 2- and 4-mm ammonium nitrate. These results provide useful information on how to manage fertility to optimize nodulation and show suppression of nodule activity caused by N fertilization can be temporary if excess N is leached from the root zone.

Nodule-like Structures Induced on the Roots of Maize Seedlings by the Addition of Synthetic Auxin 2,4-D and its Effects on Growth and Yield

Induction of para-nodules on maize contributed to increase in yield. The nodules on maize (Zea mays L.) cv. Kiran were induced using plant growth regulator 2,4-D (lppm) in nitrogen free Hoagland solution. Concentrations of 2,4-D greater than lppm caused stunting and death, while concentrations less than lppm has no obvious effect on seedling growth. The associative symbiosis (using Azospirillum) was noticed in the nodulated and non-nodulated maize plants. The presence of nitrogenase activity (acetylene reduction assay) and leghemoglobin was noticed in the plants treated with Azospirillum either alone or with nodulated plants, but higher activity was noticed in the nodulated Azospirillum treated plants. The 2,4-D induced nodule like structures provided niche for the establishment of the bacteria, thereby enhancing nitrogen fixation in the nodulated roots. These results confirmed the nitrogenase activity of Azospirillum brasilense in association with the 2,4-D induced nodules of maize. After transplanting in the pots, it was noticed that nodulated and Azospirillum treated plants showed higher chlorophyll content in leaf, enhanced NR activity leading to higher yield as compared to controlled plants (non-nodulated). The nodulated plants treated with Azospirillum had higher physiological activities as compared to only Azospirillum treated plants. Thus, the gramineous plants are potentially able to create a symbiosis with diazotropic bacteria, which colonize para-nodule tissue intracellularly, promoting a higher level of N2 fixation for the better growth and development of the plants.

Comparison of diazotroph community structure in Lyngbya sp. and Microcoleus chthonoplastes dominated microbial mats from Guerrero Negro, Baja, Mexico

The nitrogenase activity and phylogenetic diversity of nitrogen fixing microorganisms in several different cyanobacterial mat types from Guerrero Negro, Baja California, Mexico were investigated by acetylene reduction assay, and by amplification and sequencing of the nitrogenase nifH gene. Acetylene reduction assays performed on a Lyngbya sp. and two Microcoleus chthonoplastes dominated microbial mats showed a typical diel pattern of nitrogenase activity in these mats. The highest rates of activity were found at night, with 40 and 37 μmol C 2H 4 m −2 h −1 measured in the Microcoleus mats, and 9 μmol C 2H 4 m −2 h −1 in the Lyngbya mat. Nitrogenase sequences were obtained that clustered with sequences from cyanobacteria, γ-Proteobacteria, and cluster 3 of nifH. In addition, novel and divergent sequences were also recovered. The composition of nifH sequence types recovered differed between the Lyngbya and Microcoleus mats. Interestingly, nifH sequences belonging to filamentous cyanobacteria were absent in most mat samples even though both mats were dominated by filamentous cyanobacteria. nifH sequences clustering with those of unicellular cyanobacteria were found, some of which were virtually identical to the nifH sequence from Halothece sp. MPI96P605, which had previously been isolated from the mat. In manipulation experiments, the Lyngbya and Microcoleus mats were allowed to re-colonize a cleared surface. In these developing mats, nifH sequences not previously observed in the mats were discovered. Our results showed that organisms capable of N 2 fixation were present in N 2 fixing mats, that the composition of the N 2 fixing communities differs between mats, and that filamentous cyanobacterial diazotrophs may not have a large role in the early stages of mat development.

Estimating N2 fixation in two species of Alnus in interior Alaska using acetylene reduction and 15N2 uptake

:In interior Alaskan boreal forests two species of alder, Alnus tenuifolia and Alnus crispa, represent keystone species in floodplain and upland landscapes, respectively, due to the ability of these plants to form symbiotic associations with the nitrogen-fixing actinomycete, Frankia. It is believed that as much as 70% of the nitrogen (N) accumulated during the 200-y successional development of these forests is derived through atmospheric fixation by these species. Estimates of gross N inputs in these and many other ecosystems have traditionally utilized the acetylene reduction assay (ARA), which requires a conversion factor of the ratio of acetylene to N2 reduced by nitrogenase, the primary enzyme. Despite the fact that small variations in the reduction ratio can substantially influence estimates of N inputs, few studies have investigated how it varies spatially and temporally. The present study sought to 1) determine this conversion factor for both species of alder in situ by calibration of the ARA against a 15N2 uptake method we developed for field use and 2) determine whether the conversion factor varied with the successional stage in which the alders occurred. Averaged across all plants, the ratio of acetylene to N2 reduced was significantly greater in A. crispa. Significant differences in the value of the conversion factor were observed between early succession and later (mid and late) successional sites for both species. Such differences were also observed among replicate sites within and among stages. However, these stage and site differences may also be due to seasonal effects, which could not be controlled for with our design. Specific acetylene reductase activity (SARA) was only correlated with 15N2 uptake for early successional sites measured early in the growing season, when N2-fixation rates were lowest and the conversion factor was closest to the theoretical value of 4. A significant negative correlation was found between the conversion factor value and the rate of enzyme activity as determined by the 15N2 uptake method. Two hypotheses are proposed to explain this result: 1) that it is due to changes in the kinetic properties of nitrogenase at high levels of enzyme activity, resulting in an increased affinity of the enzyme for N2 relative to C2H2, and 2) that the concentration of C2H2 used in our ARA was insufficient to saturate nitrogenase.

Dinitrogen fixation and release of ammonium and dissolved organic nitrogen by Trichodesmium IMS101

Two methods used to measure dinitrogen (N 2 ) fixation (acetylene reduction and 15 N 2 uptake) often result in different N 2 fixation rates. Part of the discrepancy may arise from the observation that Trichodesmium can release a fraction of their recently fixed N 2 as dissolved organic nitrogen (DON) and/or ammonium (NH 4 + ). To resolve outstanding issues regarding N 2 fixation and the production of dissolved combined nitrogen (N) by Trichodesmium, we conducted a comprehensive analysis of N 2 fixation and the production of DON and NH 4 + in cultures of Trichodesmium IMS101. We performed 15 N 2 uptake experiments in parallel with acetylene (C 2 H 2 ) reduction assays, and measured production of 15 NH 4 + and DO 15 N from 15 N 2 , and 15 NH 4 + uptake and regeneration by isotope dilution. Four main results are highlighted. First, 15 N 2 uptake appears to provide a better approximation of net N-specific growth rates than N 2 fixation estimates made using C 2 H 2 reduction. Second, the C 2 H 2 reduction method provides a closer approximation of gross N 2 fixation. Third, simultaneous measurements of relevant N pools and pathways by several methods enabled us to rigorously evaluate deviations from theoretical conversion factors and to interpret the basis for those deviations. Our results suggest that a conversion ratio (mol C 2 H 2 reduced: mol N 2 reduced to PON, ammonium and DON) of 4:1 may be more appropriate for total N 2 fixation. Fourth, the difference between estimates of gross N 2 fixation, made using the C 2 H 2 reduction technique, and net 15 N 2 uptake into particulate N may be a good indicator of N release from N 2 fixation.

Rhizobium , Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.

Nodulation of Mimosa spp. by the beta-proteobacterium Ralstonia taiwanensis.

Several beta-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.

Nitrogen fixation in the activated sludge treatment of thermomechanical pulping wastewater: effect of dissolved oxygen

N-ViroTech, a novel technology which selects for nitrogen-fixing bacteria as the bacteria primarily responsible for carbon removal, has been developed to treat nutrient limited wastewaters to a high quality without the addition of nitrogen, and only minimal addition of phosphorus. Selection of the operating dissolved oxygen level to maximise nitrogen fixation forms a key component of the technology. Pilot scale activated sludge treatment of a thermomechanical pulping wastewater was carried out in nitrogen-fixing mode over a 15 month period. The effect of dissolved oxygen was studied at three levels: 14% (Phase 1), 5% (Phase 2) and 30% (Phase 3). The plant was operated at an organic loading of 0.7-1.1 kg BOD5/m3/d, a solids retention time of approximately 10 d, a hydraulic retention time of 1.4 d and a F:M ratio of 0.17-0.23 mg BOD5/mg VSS/d. Treatment performance was very stable over the three dissolved oxygen operating levels. The plant achieved 94-96% BOD removal, 82-87% total COD removal, 79-87% soluble COD removal, and >99% total extractives removal. The lowest organic carbon removals were observed during operation at 30% DO but were more likely to be due to phosphorus limitation than operation at high dissolved oxygen, as there was a significant decrease in phosphorus entering the plant during Phase 3. Discharge of dissolved nitrogen, ammonium and oxidised nitrogen were consistently low (1.1-1.6 mg/L DKN, 0.1-0.2 mg/L NH4+-N and 0.0 mg/L oxidised nitrogen). Discharge of dissolved phosphorus was 2.8 mg/L, 0.1 mg/L and 0.6 mg/L DRP in Phases 1, 2 and 3 respectively. It was postulated that a population of polyphosphate accumulating bacteria developed during Phase 1. Operation at low dissolved oxygen during Phase 2 appeared to promote biological phosphorus uptake which may have been affected by raising the dissolved oxygen to 30% in Phase 3. Total nitrogen and phosphorus discharge was dependent on efficient secondary clarification, and improved over the course of the study as suspended solids discharge improved. Nitrogen fixation was demonstrated throughout the study using an acetylene reduction assay. Based on nitrogen balances around the plant, there was a 55, 354 and 98% increase in nitrogen during Phases 1, 2 and 3 respectively. There was a significant decrease in phosphorus between Phases 1 and 2, and Phase 3 of the study, as well as a significant increase in nitrogen between Phases 2 and 3 which masked the effect of changing the dissolved oxygen. Operation at low dissolved oxygen appeared to confer a competitive advantage to the nitrogen-fixing bacteria.