Measurements Of Nitrogenase Activity Research Articles

Acetylene Reduction Assay: A Measure of Nitrogenase Activity in Plants and Bacteria.

Nitrogen is one of the most abundant elements in the biosphere, but its gaseous form is not biologically available to many organisms, including plants and animals. Diazotrophic microorganisms can convert atmospheric nitrogen into ammonia, a form that can be absorbed by plants in a process called biological nitrogen fixation (BNF). BNF is catalyzed by the enzyme nitrogenase, which not only reduces N2 to NH3 , but also reduces other substrates such as acetylene. The acetylene reduction assay (ARA) can be used to measure nitrogenase activity in diazotrophic organisms, either in symbiotic associations or in their free-living state. The technique uses gas chromatography to measure the reduction of acetylene to ethylene by nitrogenase in a simple, quick, and inexpensive manner. Here, we demonstrate how to: prepare nodulated soybean plants and culture free-living Azospirillum brasilense for the ARA, use the gas chromatograph to detect the ethylene formed, and calculate the nitrogenase activity based on the peaks generated by the chromatograph. The methods shown here using example organisms can be easily adapted to other nodulating plants and diazotrophic bacteria. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acetylene reduction assay in root nodules Basic Protocol 2: Acetylene reduction assay using diazotrophic bacteria Basic Protocol 3: Calculation of nitrogenase activity Support Protocol 1: Production of acetylene from calcium carbide Support Protocol 2: Calibration of the gas chromatograph Support Protocol 3: Total protein quantification.

Dry-hot stress significantly reduced the nitrogenase activity of epiphytic cyanolichen

Nitrogen (N) fixed by epiphytic cyanolichens (i.e. lichens that contain cyanobacterial symbionts) is thought to be the most important resource of this nutrient in some natural forest ecosystems. Although a great deal of work has been carried out to evaluate the biomass of this group as well as its contribution to ecosystem N budgets, empirical studies are needed to confirm the N input responses by cyanolichens under climate change conditions (dry-hot stress) as well as to determine the factors that control this process. We simulated climate change conditions by transplanting Lobaria retigera, a common cyanolichen in the area, to lower elevations, and measured nitrogenase activity in response to warmer and drier conditions. In addition, we conducted a series of laboratory and greenhouse experiments to determine the dominant factors influencing nitrogenase activity in this species. The results of this study show that mean annual nitrogenase activity at the higher site was 1.5 and 2.4 times that at the simulated warmer and drier (middle and lower) sites, respectively. Combining laboratory experimental conclusions, we show that thallus water content is a key factor determining the nitrogenase activity of L. retigera in early transplantation while insufficient carbon storage resulting from a combination of warming and desiccation was likely responsible for reducing nitrogenase activity in later months of the transplant experiment. The results of this study imply that the negative impact of climate change (dry-hot stress) on ecosystems not only impacts the distribution and growth of species, but also nutrient circles and budgets.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BackgroundCyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.ResultsBy combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.ConclusionsThis investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.

Symbiotic activity of pea (Pisum sativum) after application of Nod factors under field conditions.

Growth and symbiotic activity of legumes are mediated by Nod factors (LCO, lipo-chitooligosaccharides). To assess the effects of application of Nod factors on symbiotic activity and yield of pea, a two-year field experiment was conducted on a Haplic Luvisol developed from loess. Nod factors were isolated from Rhizobium leguminosarum bv. viciae strain GR09. Pea seeds were treated with the Nod factors (10−11 M) or water (control) before planting. Symbiotic activity was evaluated by measurements of nitrogenase activity (acetylene reduction assay), nodule number and mass, and top growth by shoot mass, leaf area, and seed and protein yield. Nod factors generally improved pea yield and nitrogenase activity in the relatively dry growing season 2012, but not in the wet growing season in 2013 due to different weather conditions.

Occurrence of benthic microbial nitrogen fixation coupled to sulfate reduction in the seasonally hypoxic Eckernförde Bay, Baltic Sea

Abstract. Despite the worldwide occurrence of marine hypoxic regions, benthic nitrogen (N) cycling within these areas is poorly understood and it is generally assumed that these areas represent zones of intense fixed N loss from the marine system. Sulfate reduction can be an important process for organic matter degradation in sediments beneath hypoxic waters and many sulfate-reducing bacteria (SRB) have the genetic potential to fix molecular N (N2). Therefore, SRB may supply fixed N to these systems, countering some of the N lost via microbial processes, such as denitrification and anaerobic ammonium oxidation. The objective of this study was to evaluate if N2 fixation, possibly by SRB, plays a role in N cycling within the seasonally hypoxic sediments from the Eckernförde Bay, Baltic Sea. Monthly samplings were performed over the course of one year to measure nitrogenase activity (NA) and sulfate reduction rates, to determine the seasonal variations in bioturbation (bioirrigation) activity and important benthic geochemical profiles, such as sulfur and N compounds, and to monitor changes in water column temperature and oxygen concentrations. Additionally, at several time points, the active N-fixing community was examined via molecular tools. Integrated rates of N2 fixation (approximated from NA) and sulfate reduction showed a similar seasonality pattern, with highest rates occurring in August (approx. 22 and 880 nmol cm−3 d−1 of N and SO42−, respectively) and October (approx. 22 and 1300 nmol cm−3 d−1 of N and SO42− respectively), and lowest rates occurring in February (approx. 8 and 32 nmol cm−3 d−1 of N and SO42−, respectively). These rate changes were positively correlated with bottom water temperatures and previous reported plankton bloom activities, and negatively correlated with bottom water oxygen concentrations. Other variables that also appeared to play a role in rate determination were bioturbation, bubble irrigation and winter storm events. Molecular analysis demonstrated the presence of nifH sequences related to two known N2 fixing SRB, namely Desulfovibrio vulgaris and Desulfonema limicola, supporting the hypothesis that some of the nitrogenase activity detected may be attributed to SRB. Overall, our data show that Eckernförde Bay represents a complex ecosystem where numerous environmental variables combine to influence benthic microbial activities involving N and sulfur cycling.

Soybean nodulation and nitrogen fixation in response to soil compaction and surface straw mulching

Soil compaction affects nodulation and nitrogen fixation of soybean ( Glycine max (L.) Merr.). The effects of compaction can be mediated by straw mulch applied at soil surface. To test these hypotheses a field experiment was carried out in 2008 on a Haplic Luvisol (south-eastern Poland). We grew soybean in treatments with different soil compaction levels: non-compacted (NC), moderately compacted (MC) (3 tractor passes) and strongly compacted soil (SC) (5 tractor passes). Each compaction level had treatments without and with surface chopped straw mulch at the rate of 0.5 kg m −2. Nodulation was evaluated by measurements of nodule-size distribution, number, weight, and diameter of nodules and symbiotic nitrogen fixation – by measurement of nitrogenase activity, nitrogen content in leaves, protein content in seeds and seed yield. Soil measurements included bulk density and nitrogen content in the form of N-NO 3 and N-NH 4. In general, nitrogenase activity decreased with increasing soil compaction level. At every compaction level, the nitrogenase activity was higher in the mulched when compared to the unmulched soil. Soil compaction and mulching enhanced the contribution of large nodules, those greater than 0.41 cm and dry weight of individual nodules. Total nodule number and weight decreased in sequence: moderately, strongly and non-compacted soil. Mulch influenced nitrogenase activity and nodulation to a higher extent than compaction. An optimum soybean response in terms of seed and protein yields was observed under mulched MC and the worst – under unmulched SC. Both N content in leaves and nitrate content in soil were lower in mulched than in unmulched soil, irrespective of compaction. Bulk density and N-NO 3 increased with soil compaction level and N-NH 4 remained nearly unchanged. Therefore, our results supported the hypotheses that soil compaction affects nodulation and nitrogen fixation, and that compaction can be mediated by straw mulch. Further studies are needed to assess variability related with different soil and weather conditions.

Nitrogen metabolism and seed composition as influenced by foliar boron application in soybean

The physiological effects of foliar boron application (FB) on nitrogen metabolism and seed composition have not been well established in soybean [(Glycine max(L.)Merr.)]. Therefore, the effect of FB on nitrogen metabolism and seed composition was investigated. Nitrate assimilation was evaluated by measuring nitrate reductase activity (NRA) and nitrogen fixation was evaluated by measuring nitrogenase activity and natural abundance of 15N/14N. NRA were significantly (P ≤ 0.05) higher in plants that received FB than the control plants. Higher rate of FB (One application of four times of commercial rate) inhibited nitrogen fixation as measured by natural abundance of 15N/14N ratio, but increased NRA. The higher activities of NR and nitrogenase by FB were accompanied with a higher B concentration in leaves. The significant (P < 0.0001) enrichment of 15N/14N, accompanied with a higher rate of FB, suggested a possible mechanism where nitrate assimilation may compensate for the decrease in nitrogen fixation. FB increased seed protein by 13.7% and oleic acid by 30.9% compared to the control plants. This alteration was accompanied by a higher B concentration in leaves and seed. The results suggest that FB affects nitrogen metabolism and alters seed compositions, especially protein and unsaturated fatty acids.

Potential nitrogen fixation activity of different aged biological soil crusts from rehabilitated grasslands of the hilly Loess Plateau, China

Biological soil crusts (biocrusts) cover up to 60–70% of the soil surface in grasslands rehabilitated during the “Grain for Green” project implemented in the hilly Loess Plateau region in 1999. As biocrusts fix nitrogen (N), they are an important part of restoring soil fertility. We measured nitrogenase activity (NA) in biocrusts from sites rehabilitated at six different time periods to estimate 1) the effects of moisture content and temperature on NA in biocrusts of different ages and 2) the potential N contribution from biocrusts to soils and plants in this region. Results show that NA in the biocrusts was mostly controlled by the species composition, as the activity of biocrusts dominated by free-living soil cyanobacteria was significantly higher than that of moss-dominated biocrusts. Nitrogenase activity was also influenced by soil moisture content and ambient temperature, with a significant decline in activity when moisture levels were decreased to 20% field water-holding capacity. The optimal temperature for NA was 35–40 °C and 30–40 °C for cyanobacteria- and moss-dominated biocrusts, respectively. Biocrust fixed N is likely an important source of N in this ecosystem, as we estimated annual potential N inputs per hectare in these grasslands to be up to 13 kg N ha −1 and 4 kg N ha −1 for cyanobacteria- and moss-dominated biocrusts, respectively.

Protection of free-living nitrogen-fixing bacteria within the soil matrix

Habitat based protection gives soil microbial communities the ability to withstand unfavourable environmental periods. Soils differ in their ability to support and maintain microbial functions through their variation in physical and chemical properties. This paper explores the use of biocidal treatments to assess the ability of soils to protect N 2-fixing communities from adverse conditions. The acetylene reduction assay was used to measure nitrogenase activity by free-living bacteria in soil exposed to additional C, and biocidal treatments (steam or CHCl 3). N 2-fixing microbial communities in soils with higher clay contents (∼50%) withstood fumigation treatment, demonstrated through acetylene reduction activity and a nifH PCR response, whereas those in sandy soils failed to survive. Based on this study and other research on soil aggregates, it was concluded that microbial survival in different soils was related to the ability of aggregates to provide protection against biocidal exposure within the soil matrix. This has consequences for the amount and duration N 2-fixing activities in soils exposed to adverse environmental conditions.

Interactive effects of elevated CO2, phosphorus deficiency, and soil drought on nodulation and nitrogenase activity in Alnus hirsuta and Alnus maximowiczii

We examined the interactive effects of elevated CO2, soil phosphorus (P) availability, and soil drought on nodulation, nitrogenase activity, and biomass allocation in Alnus hirsuta and Alnus maximowiczii. Potted seedlings were grown in either ambient or elevated CO2 (36 Pa and 72 Pa CO2), with different levels of P (7.7 and 0.77 mgP pot−1 week−1 for high-P and low-P, respectively) and water supply in a natural daylight phytotron. Measurements of nitrogenase activity by an acetylene reduction assay failed to reveal significant effects of the treatments in any species. In high-P, nodule biomass increased under elevated CO2 and decreased under drought. In low-P, nodule biomass decreased substantially compared to high-P, but the effect of elevated CO2 on nodule biomass was unclear. Soil drought increased the partitioning of biomass into nodules, especially in A. hirsuta. These results suggest that with high P availability, elevated CO2 could promote N2 fixation by increasing nodule biomass even under drought. On the other hand, if soil P is limiting, elevated CO2 may not enhance N2 fixation because of the suppression of growth.

Acclimation of Trichodesmium erythraeum ISM101 to high and low irradiance analysed on the physiological, biophysical and biochemical level

As the nonheterocystous diazotrophic cyanobacterium Trichodesmium lives both at the ocean surface and deep in the water column, it has to acclimate to vastly different irradiances. Here, we investigate its strategy of light acclimation in several ways. In this study, we used spectrally resolved fluorescence kinetic microscopy to investigate the biophysics of photosynthesis in individual cells, analysed cell extracts for pigment and phycobiliprotein composition, measured nitrogenase activity and the abundance of key proteins, and assayed protein synthesis/degradation by radioactive labelling. After acclimation to high light, Trichodesmium grew faster at 1000 micromol m(-2) s(-1) than at 100 micromol m(-2) s(-1). This acclimation was associated with decreasing cell diameter, faster protein turnover, the down-regulation of light-harvesting pigments and the outer part of the phycobiliprotein antenna, the up-regulation of light-protective carotenoids, changes in the coupling of phycobilisomes to the reaction centres and in the coupling of individual phycobiliproteins to the phycobilisomes. The latter was particularly interesting, as it represents an as yet unreported light acclimation strategy. Only in the low light-acclimated culture and only after the onset of actinic light did phycourobilin and phycoerythrin contribute to photochemical fluorescence quenching, showing that these phycobiliproteins may become quickly (in seconds) very closely coupled to photosystem II. This fast reversible coupling also became visible in the nonphotochemical changes of the fluorescence quantum yield.

Effects of ultraviolet radiation on productivity and nitrogen fixation in the Cyanobacterium, Anabaena sp. (Newton’s strain)

The biological effects of ultraviolet radiation (UVR; 290–400 nm), especially the UV-B (320–400 nm) component of the spectrum, include both direct and indirect effects on many cellular processes. In cyanobacteria both photosynthesis and nitrogen fixation can be affected directly by UVR, and indirectly by UVR through the production of reactive oxygen species (ROS). For the heterocystous cyanobacterium, Anabaena sp. (Newton’s strain), exposure to UVR causes a significant decline in the quantum yields of photosystem II (PSII) fluorescence and maximum productivity despite an increase in UVR absorbing compounds, mycosporine-like amino acids (MAAs), in those cells exposed to UVR. Concurrent with these observations are significant increases in the activities of superoxide dismutase indicative of an increase in the level of oxidative stress in cells exposed to UVR. Additionally, measurements of nitrogenase activity (acetylene reduction) show a significant decrease in cyanobacteria exposed to UVR, which manifests itself as a decrease in cellular nitrogen and an increase in C:N ratios. These results show that these nitrogen-fixing cyanobacteria are particularly sensitive to UVR, both its direct and indirect effects. The effects of UVR reported here add to the increasing evidence that UVR effects on this important group of prokaryotes could affect the input of new nitrogen, and the biogeochemical cycling of this essential macronutrient in terrestrial, marine, and freshwater habitats.

Carbon Dioxide Effects on Heterotrophic Dinitrogen Fixation in a Temperate Pine Forest

Increased net primary productivity (NPP) under elevated atmospheric CO2 requires additional N inputs to sustain C sequestration. We hypothesized that heterotrophic N2 fixation would be stimulated by enhanced litter production under elevated CO2, thus augmenting N availability to plants. To test if N2 fixation is limited by organic substrates alone or in combination with nutrients required for the nitrogenase enzyme, we measured nitrogenase activity (acetylene reduction) in laboratory incubations with water, nutrient, and O2 manipulations. Response of N2 fixation to water, glucose, P, Fe, or Mo was measured under aerobic and anaerobic conditions in forest floor and mineral soil samples from the Duke Forest, NC. Potential nitrogenase activity in forest floor and mineral soil from the Duke Forest Free Air CO2 Enrichment (FACE) site were measured to determine if elevated CO2 enhances N2 fixation. In homogenized slurries with glucose additions, nitrogenase activity was 2 and 400 times greater than controls in organic and mineral soils, respectively (P &lt; 0.01). In laboratory studies, water additions increased N2 fixation 25‐fold in intact soil cores (P &lt; 0.01). Additions of nutrients alone or in combination with C and water did not consistently stimulate N2 fixation in intact soil cores. We detected no CO2 effect on potential nitrogenase activity in Duke FACE soil. Since heterotrophic N2 fixation is not stimulated in temperate pine forests under elevated CO2, additional N assimilation by trees will require increased acquisition of endogenous N, such as increased nutrient use efficiency or enhanced root exploration of the soil.

Effect of weed management on weeds, and on the nodulation, nitrogenase activity, growth and yield of pea (Pisum sativum)

Effects of one pre-emergence herbicide (terbutryn/terbuthylazine) and one post-emergence herbicide (bentazone) along with unweeded and hand-weeded controls on weeds and on the nodulation, nitrogenase activity, nitrogen content, growth and yield of pea (Pisum sativum) were studied. Terbutryn/terbuthylazine was applied pre-emergence @ 1.40, 2.80 and 5.60 kg/hawhereas bentazone was sprayed 6 weeks after sowing @ 1.44, 2.88 and 5.76 kg/h. Terbutryn/terbuthylazine controlled all the weeds very effectively, whereas bentazone did not control some weeds such as Polygonum aviculare, Poa annua and Elymus repens. The herbicides decreased the number of nodules, the dry weight of nodules, the nitrogenase activity, the shoot dry weight, the nitrogen content in the straw and seeds, and the seed yield of peas, the effects generally being higher at higher rates of application. The adverse effects of herbicides on these parameters might be due to their effects on plant growth, as both the herbicides are known to adversely affect photosynthesis. Nitrogenase activity did not correlate well with plant-N content or shoot dry weight. However, there was a strong relationship between plant biomass and plant-N content, which suggests that researchers can rely on these parameters for studying the effects of treatments on nitrogen fixation, rather than measuring nitrogenase activity.

Simulated Glyphosate Drift Influences Nitrate Assimilation and Nitrogen Fixation in Non-glyphosate-Resistant Soybean

Nontarget injury from glyphosate drift is a concern among growers using non-glyphosate-resistant (non-GR) cultivars. The effects of glyphosate drift on nitrate assimilation and nitrogen fixation potential, nodule mass, and yield of non-GR soybean were assessed in a field trial at Stoneville, MS. A non-GR soybean cultivar 'Delta Pine 4748S' was treated with glyphosate at 12.5% of use rate of 0.84 kg of active ingredient/ha at 3 (V2), 6 (V7), and 8 (R2, full bloom) weeks after planting (WAP) soybean to simulate glyphosate drift. Untreated soybean was used as a control. Soybeans were sampled weekly for 2 weeks after each glyphosate treatment to assess nitrate assimilation and N2 fixation potential. Nitrate assimilation was assessed using in vivo nitrate reductase assay in leaves, stems, roots, and nodules. Nitrogen fixation potential was assessed by measuring nitrogenase activity using the acetylene reduction assay (ARA). Nitrogen content of leaves, shoots, and seed and soybean yield were also determined. In the first sampling date (4 WAP), glyphosate drift caused a significant decrease in NRA in leaves (60%), stems (77%), and nodules (50%), with no decrease in roots. At later growth stages, NRA in leaves was more sensitive to glyphosate drift than stems and roots. Nitrogenase activity was reduced 36-58% by glyphosate treatment at 3 or 6 WAP. However, glyphosate treatment at 8 WAP had no effect on nitrogenase activity. Nitrogen content was affected by glyphosate application only in shoots after the first application. No yield, seed nitrogen, protein, or oil concentration differences were detected. These results suggest that nitrate assimilation and nitrogen fixation potential were significantly reduced by glyphosate drift, with the greatest sensitivity early in vegetative growth. Soybean has the ability to recover from the physiological stress caused by glyphosate drift.

Nitrogen fixation in coarse woody debris of Thuja plicata and Tsuga heterophylla forests on northern Vancouver Island

Asymbiotic nitrogenase activity in coarse woody debris was measured using the acetylene reduction assay under ambient conditions in three different stand ages (5, 53, and 88 years old) of an unmanaged second-growth Tsuga heterophylla (Raf.) Sarg. – Abies amabilis (Dougl. ex Loud.) Dougl. ex J. Forbes forest type and a Thuja plicata Donn. ex D. Don – Tsuga heterophylla old-growth forest on northern Vancouver Island, British Columbia, Canada. Four different decay classes of coarse woody debris, different species in the early decay stages, and sapwood and heart wood were sampled separately. Mean nitrogenase activity ranged between 1.3 and 19.5 nmol C2H4·d–1·(g dry mass)–1, with an overall mean of 5.7. High variability of the activity rates between logs and within logs was observed in all four stands. Mean activity rates were, in most cases, significantly different between decay classes, with generally increasing nitrogenase activity with the progress of decay. Moisture content of the samples was a good predictor of nitrogenase activity and could explain differences between decay classes. Only minor differences in nitrogenase activity were found between the different stands. Estimates of nitrogen fixation ranged from 1.0 to 2.1 kg N·ha–1·year–1, the magnitude of these values depending more on the mass of coarse woody debris substrate available for asymbiotic nitrogen-fixing bacteria (103–158 t·ha–1 in this study) than on differences in nitrogenase activity rates. The measured nitrogenase activity and the resultant estimates of nitrogen fixation are among the highest values reported in the literature.

NITROGEN CYCLING MEDIATED BY BIOLOGICAL SOIL CRUSTS AND ARBUSCULAR MYCORRHIZAL FUNGI

Plants in terrestrial systems obtain nitrogen primarily by root uptake from soil, yet current models of ecosystem nitrogen cycling assume little interaction of plant roots with the soil microbial community. The objective of this study was to determine how soil microorganisms in biological soil crusts interact with herbaceous plants for nitrogen in a pyrogenic ecosystem in Florida (USA), using a combination of natural abundance and tracer isotope methods. I demonstrate that plant nitrogen is strongly affected by interactions with mycorrhizal fungi and biological soil crusts. Measurements of nitrogenase activity indicated a high capacity of crusts to fix nitrogen that dramatically decreased after disturbance. Natural abundance of δ15N differed between arbuscular mycorrhizal and nonmycorrhizal herbs. When a small amount of enriched nitrogen was used as a tracer, crusts initially captured a greater amount of the 15N that was added compared to plants, with some turnover of nitrogen from the crusts to plants ob...

Effect of elevated carbon dioxide in the root atmosphere on nitrogenase activity in three actinorhizal plant species

In wet soils, nitrogen-fixing root nodules are subjected to elevated CO2. Only a few studies have examined the effect of elevated CO2 on nitrogenase activity, and the results have been mixed. We examined intact black alder (Alnus glutinosa (L.) Gaertn.) and sweet gale (Myrica gale L.) seedlings and field-collected speckled alder (Alnus incana ssp. rugosa (Du Roi) Claus.) nodules to clarify the effects of elevated CO2. Nitrogenase activity was measured via acetylene reduction in an open, flow-through system. We found that repeated measurements of the peak rate of nitrogenase activity, the only reliable measure of nitrogenase activity, could be made on the same plant via 150-s exposures to acetylene separated by 20 min without acetylene. Our results for elevated CO2 consistently showed that it had little effect on nitrogenase activity at low concentrations and increasingly inhibited nitrogenase activity as the CO2 concentration increased. In black alder, 0.5 kPa CO2 had little effect, whereas 3.0 kPa CO2 reduced nitrogenase activity 31–35%. Sweet gale, was less sensitive to elevated CO2 and was unaffected by 1.5 kPa CO2. Black alder grown with the roots, but not the shoots, in 1.3 kPa CO2 showed only minimal acclimation to elevated CO2.Key words: acetylene reduction technique, actinorhizal plants, Alnus, carbon dioxide, Myrica gale, nitrogen fixation.

Nodulation Status and Nitrogenase Activity of Some Legume Tree Species in Bangladesh

Summary A study was conducted to observe the nodulation status and measure nitrogenase activity of some important legume tree species in nursery. Of the thirteen species surveyed eight belong to Mimoceae, two Papilionaceae and three Caesalpiniaceae. All the species in Mimo-ceae and Papilionaceae were found nodulated whereas all the members of Caesalpiniaceae were non-nodulated. Among nodulated seedlings, highest number of nodules per seedling was recorded in Leucaena leucocephala (82) followed by Acacia auriculiformis (55), Acacia mangium (52), Albizia lebbeck (46), A. procera (41), Dalbergia sissoo (32) and Acacia catechu (29). Nitrogenase activity was highest in L. leucocephla (4913.59 nmole C2H4 h−1) followed by Albizia procera (2080 nmole C2H4 h−1). Seedling height, nodule fresh weight, root fresh weight and nitrogenase activity (per nodule per h, per gram nodule fresh weight per h, per gram root fresh weight per h and per gram root dry weight per h) were also highest in L. leucocephala.

Changes in Asymbiotic, Heterotrophic Nitrogen Fixation on Leaf Litter of Metrosideros polymorpha with Long-Term Ecosystem Development in Hawaii

We measured nitrogenase activity (acetylene reduction) of asymbiotic, heterotrophic, nitrogen-fixing bacteria on leaf litter from the tree Metrosideros polymorpha collected from six sites on the Hawaiian archipelago. At all sites M. polymorpha was the dominant tree, and its litter was the most abundant on the forest floor. The sites spanned a soil chronosequence of 300 to 4.1 million y. We estimated potential nitrogen fixation associated with this leaf litter to be highest at the youngest site (1.25 kg ha-1 y-1), declining to between 0.05 and 0.22 kg ha-1 y-1 at the oldest four sites on the chronosequence. To investigate how the availability of weathered elements influences N fixation rates at different stages of soil development, we sampled M. polymorpha leaf litter from complete, factorial fertilization experiments located at the 300-y, 20,000-y and 4.1 million–y sites. At the youngest and oldest sites, nitrogenase activity on leaf litter increased significantly in the plots fertilized with phosphorus and “total” (all nutrients except N and P); no significant increases in nitrogenase activity were measured in leaf litter from treatments at the middle-aged site. The results suggest that the highest rates of N fixation are sustained during the “building” or early phase of ecosystem development when N is accumulating and inputs of geologically cycled (lithophilic) nutrients from weathering are substantial.