Symbiotic unicellular cyanobacteria fix nitrogen in the Arctic Ocean.

Biological dinitrogen (N2) fixation is an important source of nitrogen (N) in low-latitude open oceans. The unusual N2-fixing unicellular cyanobacteria (UCYN-A)/haptophyte symbiosis has been found in an increasing number of unexpected environments, including northern waters of the Danish Straight and Bering and Chukchi Seas. We used nanoscale secondary ion mass spectrometry (nanoSIMS) to measure 15N2 uptake into UCYN-A/haptophyte symbiosis and found that UCYN-A strains identical to low-latitude strains are fixing N2 in the Bering and Chukchi Seas, at rates comparable to subtropical waters. These results show definitively that cyanobacterial N2 fixation is not constrained to subtropical waters, challenging paradigms and models of global N2 fixation. The Arctic is particularly sensitive to climate change, and N2 fixation may increase in Arctic waters under future climate scenarios

Molecular evidence for sediment nitrogen fixation in a temperate New England estuary

Primary production in coastal waters is generally nitrogen (N) limited with denitrification outpacing nitrogen fixation (N2-fixation). However, recent work suggests that we have potentially underestimated the importance of heterotrophic sediment N2-fixation in marine ecosystems. We used clone libraries to examine transcript diversity of nifH (a gene associated with N2-fixation) in sediments at three sites in a temperate New England estuary (Waquoit Bay, Massachusetts, USA) and compared our results to net sediment N2 fluxes previously measured at these sites. We observed nifH expression at all sites, including a site heavily impacted by anthropogenic N. At this N impacted site, we also observed mean net sediment N2-fixation, linking the geochemical rate measurement with nifH expression. This same site also had the lowest diversity (non-parametric Shannon = 2.75). At the two other sites, we also detected nifH transcripts, however, the mean N2 flux indicated net denitrification. These results suggest that N2-fixation and denitrification co-occur in these sediments. Of the unique sequences in this study, 67% were most closely related to uncultured bacteria from various marine environments, 17% to Cluster III, 15% to Cluster I, and only 1% to Cluster II. These data add to the growing body of literature that sediment heterotrophic N2-fixation, even under high inorganic nitrogen concentrations, may be an important yet overlooked source of N in coastal systems

Input of nitrogen from N2 fixation to northern grasslands

Forage legumes form N2-fixing symbioses with rhizobia and may thus make substantial contributions to the N pool in grasslands. However, to optimize their use as sources of N, it is important to elucidate the effects of management factors that influence their N2 fixation rates, and to develop convenient methods for measuring N2 fixation quickly and reliably. An analysis of published data on N2 fixation in the field showed that lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), and white clover (T. repens L.) grown in mixtures with grasses derived most of their N from N2 fixation, irrespective of geographic location and management practices – and despite large inter-annual variations in legume dry matter yield (kg ha-1 year-1). Consequently, there were strong correlations between legume dry matter yield and amounts of N2 fixed (kg N ha-1 year-1), which can be used very simply to obtain estimates of N2 fixation in these legumes. In experimental grassland plots where the species richness of neighbouring vegetation was varied, alsike clover (T. hybridum L.), red clover, and white clover consistently derived at least half of their N from N2 fixation, measured by the 15N natural abundance (NA) method using three different reference plants. This method is sensitive to the degree of discrimination against 15N in the N2-fixing plant (B value) and the choice of reference plant. B values were therefore established for each of the three clover species in symbioses with different Scandinavian Rhizobium leguminosarum bv. trifolii genotypes. In red clover, reductions following cutting in the activity of the N2-fixing enzyme, nitrogenase, and the rate of shoot regrowth were dependent on the cutting height. The recovery in nitrogenase activity after cutting followed the rate of leaf area increment, which confirms the correlation between N2 fixation and growth found in field experiments. The results of the work underlying this thesis show that perennial forage legumes growing in grasslands are highly dependent on N2 fixation. Awareness of this should facilitate the development of resource-efficient management regimes for northern grasslands

Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3

Frankia is a representative genus of nitrogen-fixing (N2-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N2 fixation-specific structure (vesicle) and the regulation of N2 fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 candidate N2 fixation mutants. Twelve of these mutants were selected for further study, and their abilities to grow in NH3-deficient (N-) liquid media and their rates of acetylene reduction activities were evaluated. Eleven mutant strains were confirmed to lack the ability to fix N2. Five mutant strains formed significantly reduced numbers of vesicles, while some failed to form large mature vesicles. These vesicle mutants also exhibited an aberrant hyphal morphology, suggesting a relationship between vesicle differentiation and hyphal branching. Ten mutants showed significant reductions in the expression of nifE, nifH, and nifV genes under N- conditions. The genome sequencing of eight mutants identified 20 to 400 mutations. Although mutant strains N3H4 and N6F4 shared a large number of mutations (108), most were unique to each strain. Mutant strain N7C9 had 3 mutations in the nifD and nifH genes that may result in the inability to fix N2. The other mutant strains did not have any mutations in any known N2 fixation-related genes, indicating that they are novel N2 fixation mutants

N2-fixation and residual N effect of four legume species and four companion grass species

Inclusion of forage legumes in low-input forage mixtures improves herbage production and soil fertility through addition of nitrogen (N) from N2-fixation. The impact of different grass-legume mixtures on the N contribution of the forage mixture has rarely been investigated under comparable soil and climatic conditions. We conducted a field experiment on a sandy soil at two nitrogen levels with seven two-species forage mixtures: alfalfa, bird’s-foot trefoil, red clover, or white clover in mixture with perennial ryegrass, and white clover in mixture with meadow fescue, timothy, or hybrid ryegrass. We found high N2-fixation of more than 300 kg N ha-1 from both red clover and alfalfa even when the two mixtures received 300 kg total-N ha-1 in cattle slurry. The addition of cattle slurry N fertilizer lowered N2-fixation for white clover and red clover as expected, but for bird’s-foot trefoil and alfalfa no changes in the proportion of N derived from N2-fixation was observed. We conclude that the competition for available soil N from perennial ryegrass in mixture was an important factor for the proportion of N in alfalfa, white clover, and bird’s-foot trefoil obtained from N2-fixation. White clover had a high proportion of N derived from atmosphere for all companion grasses despite significant differences in white clover proportion. Although the perennial ryegrass-alfalfa mixture in the grass phase yielded more than twice the N from N2-fixation compared to white clover in the perennial ryegrass mixture, this did not in the following year lead to higher residual N effects of alfalfa. Both in terms of N yield in the grass phase and N yield in the subsequent spring barley red clover contributed most to the improvement of soil N fertility

From N2 fixation to N2O emission in a grass-clover mixture

In organic dairy farming, a major N input to the plant-soil system comes from biological N2 fixation by pasture legumes, but knowledge is sparse on how much of the fixed N2 is lost from the pastures as N2O. Nitrifying and denitrifying bacteria are the main contributors to the N2O production in soils. Currently, no contribution from biological N2 fixation in legume pastures is included in the national N2O inventories, partly because of uncertainties in quantifying the N2 fixation in the pastures (Mosier et al., 1998). According to the guidelines issued by The Intergovernmental Panel on Climate Change (IPCC), inventories for N2O emissions from agricultural soils should be based on the assumption that 1.25 % of added N is emitted as N2O (IPCC, 1997). The standard N2O emission factor of 1.25 % could be considerably unrepresentative for biologically fixed N2. Firstly, only a part of the fixed N is mineralised during the lifetime of the crop. Secondly, the release of inorganic N into the soil occurs slowly. A 15N2-tracer-experiment was initiated on grass-clover grown in pots. The aim was to assess: · the contribution of recently fixed N2 as a source of N2O · the translocation of N from clover to companion grass References IPCC, 1997. Greenhouse gas inventory. Reference manual. Vol. 3. Intergovernmental Panel on Climate Change. Bracknell, UK. Mosier, A. et al. 1998. Nutrient Cycling in Agroecosystems 52, 225-248

Revisiting the distribution of oceanic N₂ fixation and estimating diazotrophic contribution to marine production

Marine N2 fixation supports a significant portion of oceanic primary production by making N2 bioavailable to planktonic communities, in the process influencing atmosphere-ocean carbon fluxes and our global climate. However, the geographical distribution and controlling factors of marine N2 fixation remain elusive largely due to sparse observations. Here we present unprecedented high-resolution underway N2 fixation estimates across over 6000 kilometers of the western North Atlantic. Unexpectedly, we find increasing N2 fixation rates from the oligotrophic Sargasso Sea to North America coastal waters, driven primarily by cyanobacterial diazotrophs. N2 fixation is best correlated to phosphorus availability and chlorophyll-a concentration. Globally, intense N2 fixation activity in the coastal oceans is validated by a meta-analysis of published observations and we estimate the annual coastal N2 fixation flux to be 16.7 Tg N. This study broadens the biogeography of N2 fixation, highlights the interplay of regulating factors, and reveals thriving diazotrophic communities in coastal waters with potential significance to the global nitrogen and carbon cycles

Biomass production and N2-fixation in seven grass-legume mixtures

Inclusion of forage legumes in low-input grassland mixtures improves biomass production and soil fertility trough addition of nitrogen (N) from N2-fixation. The impacts of different mixture of legumes and companion grasses on the N production of the forage mixture have rarely been investigated under comparable soil and climatic conditions. We conducted a field experiment on a sandy soil at two nitrogen levels with seven two-species grassland mixtures: alfalfa (Medicago sativa), bird’s-foot trefoil (Lotus corniculatus), red clover (Trifolium pratense), or white clover (Trifolium repens) in mixture with perennial ryegrass (Lolium perenne), and white clover in mixture with meadow fescue (Festuca pratensis), timothy (Phleum pratense), or hybrid ryegrass (Lolium hybridum). Red clover and alfalfa fixed 400-500 kg N ha-1 and bird ’s-foot trefoil just above 100 kg N ha-1 in aboveground biomass. The white clover N fixation was affected by the companion grass species and ranged from 150 to 175 kg N ha-1. Fertilization had different effects on N2-fixation among the legumes, but also significant effects on white clover N2-fixation depending on the companion grass species

N2O emission from grass-clover swards is largely unaffected by recently fixed N2

The contribution of biologically fixed dinitrogen (N2) to the nitrous oxide (N2O) production in grasslands is unknown. To assess the contribution of recently fixed N2 as a source of N2O and the transfer of fixed N from clover to companion grass, mixtures of white clover and perennial ryegrass were incubated for 14 days in a 15N2-enriched atmosphere (0.4 atom% excess). Immediately after labelling, half of the grass-clover pots were sampled for N2 fixation determination, whereas the remaining half were examined for emission of 15N labelled N2O for another eight days using a static chamber method. Biological N2 fixation measured in grass-clover shoots and roots as well as in soil constituted 342, 38 and 67 mg N m-2 d-1 at 16, 26 and 36 weeks after emergence, respectively. The drop in N2 fixation was most likely due to a severe aphid attack on the clover component. Transfer of recently fixed N from clover to companion grass was detected at 26 and 36 weeks after emergence and amounted to 0.7 ± 0.1 mg N m-2 d-1, which represented 1.7 ± 0.3 % of the N accumulated in grass shoots during the labelling period. Total N2O emission was 91, 416 and 259 μg N2O-N m-2 d-1 at 16, 26 and 36 weeks after emergence, respectively. Only 3.2 ± 0.5 ppm of the recently fixed N2 was emitted as N2O on a daily basis, thus recently fixed N released via easily degradable clover residues appears to be a minor source of N2O

Estimating Nitrogen Fixation Rates, Importance, and Short-Term Efficiency in Small, Temperate Reservoirs Using Delta15N Techniques

Nitrogen (N2) fixation can give certain species of cyanobacteria a competitive advantage in lake and reservoir phytoplankton. These species of cyanobacteria, along with others that cannot fix N2, can form toxic compounds that impair water quality when present in high concentrations. N2 fixation rates may be substantial in small (\u3c 1.0 km2), temperate reservoirs since these systems experience thermal stratification and often nitrogen (N) limitation throughout a substantial proportion of the year. However, the effects of N2 fixation on N cycling, alleviation of short-term N limitation, and water quality are not well-understood. A mesocosm experiment and ecosystem-scale observational study were conducted to 1) determine the efficiency of N2 fixation under varying N relative to phosphorus (P) supply, 2) examine the effects of N2 fixation on autotrophic biomass accumulation and microcystin production, and 3) measure N2 fixation rates and importance to autotrophic N demand and zooplankton N assimilation. Results of the mesocosm experiment indicated that N2 fixation was increased at low N:P supply under high P. However, N2 fixation was inefficient at alleviating N limitation when fixed N was the primary source of N. Additionally, microcystin production occurred only at high N:P supply when N2 fixation was low, indicating that reducing external N inputs may have a positive effect on water quality. Results of whole-reservoir determination of N2 fixation using seston δ15N natural abundances indicated that N2 fixation rates throughout the warm season were substantial and influenced by water temperature. Annual N2 fixation rates ranged from 2.2 - 6.6 g N m-2 yr-1, and contributed up to 19% of the annual autotrophic N demand. Zooplankton were assimilating fixed N in most of the study reservoirs, representing a possible mechanism of ecosystem fixed N retention. Collectively, these results suggest that N2 fixation plays a substantial role in N cycling in small, temperate reservoirs, but likely cannot alleviate short-term N limitation