Low N2 Fixation Rates Research Articles

Highly diverse diazotrophs drive high N2 fixation rates in a shallow submarine hydrothermal system

Dinitrogen (N2) fixation provides nitrogen sources supporting active chemoautotrophic processes in submarine hydrothermal systems. Here, the diazotrophic phylogenetic diversity and N2 fixation profile were investigated in depth along a sharp biogeochemical gradient from the hydrothermal vents to ambient seawater in a shallow submarine hydrothermal system (SHS) off Kueishantao Islet, Taiwan. Diazotrophic community compositions in the SHS covered all four known nifH clusters (I, II, III, and IV) and contained both characteristics of diazotrophic communities in deep-sea hydrothermal systems and terrestrial geothermal systems. The seawater around the vents was primarily dominated by nifH phylotypes affiliated with chemolithotrophic sulfur-oxidizing and iron-reducing bacteria. In contrast, in the seawater directly above the hydrothermal vents, the nifH community markedly contained diverse heterotrophic bacteria. The analysis, which focused on amino acid composition, protein spatial structure, and the possible co-occurrence of alternative nitrogenases in the presence of molybdenum-nitrogenase, further revealed that diazotrophic nitrogenase found around the hydrothermal vents exhibits potential molecular adaptations to high temperature, low pH, and sulfur-rich conditions. The relatively low N2 fixation rates (NFRs) were observed around the vents, where high concentrations of inorganic nitrogen compounds were present. However, notably, high NFRs were detected in seawater directly above the hydrothermal vents, which were 2–5 times higher than those at the reference site. The abundant iron, along with the depletion of nitrogen nutrients potentially resulting from nitrogen assimilation and loss processes, may have led to a high iron:nitrogen ratio, inducing the observed high NFRs. The co-occurrence of diverse nifH-containing heterotrophic bacteria and high NFRs suggests that these heterotrophs may play a major role in N2 fixation in this environment. This study suggests that the SHSs may be potential hotspots for marine N2 fixation, contributing significantly to the global ocean's N2 fixation budget and warranting further investigation.

Size-fractionated N2 fixation off the Changjiang Estuary during summer.

Recent evidence has shown active N2 fixation in coastal eutrophic waters, yet the rate and controlling factors remain poorly understood, particularly in large estuaries. The Changjiang Estuary (CE) and adjacent shelf are characterized by fresh, nitrogen-replete Changjiang Diluted Water (CDW) and saline, nitrogen-depletion intruded Kuroshio water (Taiwan Warm Current and nearshore Kuroshio Branch Current), where N2 fixation may be contributed by different groups (i.e., Trichodesmium and heterotrophic diazotrophs). Here, for the first time, we provide direct measurement of size-fractionated N2 fixation rates (NFRs) off the CE during summer 2014 using the 15N2 bubble tracer method. The results demonstrated considerable spatial variations (southern > northern; offshore > inshore) in surface and depth-integrated NFRs, averaging 0.83 nmol N L-1 d-1 and 24.3 μmol N m-2 d-1, respectively. The highest bulk NFR (99.9 μmol N m-2 d-1; mostly contributed by >10 μm fraction) occurred in the southeastern East China Sea, where suffered from strong intrusion of the Kuroshio water characterized by low N/P ratio (<10) and abundant Trichodesmium (up to 10.23 × 106 trichomes m-2). However, low NFR (mostly contributed by <10 μm fraction) was detected in the CE controlled by the CDW, where NOx concentration (up to 80 μmol L-1) and N/P ratio (>100) were high and Trichodesmium abundance was low. The >10 μm fraction accounted for 60% of depth-integrated bulk NFR over the CE and adjacent shelf. We speculated that the present NFR of >10 μm fraction was mostly supported by Trichodesmium. Spearman rank correlation indicated that the NFR was significantly positively correlated with Trichodesmium abundance, salinity, temperature and Secchi depth, but was negatively with turbidity, N/P ratio, NOx, and chlorophyll a concentration. Our study suggests that distribution and size structure of N2 fixation off the CE are largely regulated by water mass (intruded Kuroshio water and CDW) movement and associated diazotrophs (particularly Trichodesmium) and nutrient conditions.

Do Nitrogen and Phosphorus Additions Affect Nitrogen Fixation Associated with Tropical Mosses?

Tropical cloud forests are characterized by abundant and biodiverse mosses which grow epiphytically as well as on the ground. Nitrogen (N)-fixing cyanobacteria live in association with most mosses, and contribute greatly to the N pool via biological nitrogen fixation (BNF). However, the availability of nutrients, especially N and phosphorus (P), can influence BNF rates drastically. To evaluate the effects of increased N and P availability on BNF in mosses, we conducted a laboratory experiment where we added N and P, in isolation and combined, to three mosses (Campylopus sp., Dicranum sp. and Thuidium peruvianum) collected from a cloud forest in Peru. Our results show that N addition almost completely inhibited BNF within a day, whereas P addition caused variable results across moss species. Low N2 fixation rates were observed in Campylopus sp. across the experiment. BNF in Dicranum sp. was decreased by all nutrients, while P additions seemed to promote BNF in T. peruvianum. Hence, each of the three mosses contributes distinctively to the ecosystem N pool depending on nutrient availability. Moreover, increased N input will likely significantly decrease BNF associated with mosses also in tropical cloud forests, thereby limiting N input to these ecosystems via the moss-cyanobacteria pathway.

Contribution of photic and aphotic N2 fixation to production in an oligotrophic sea

AbstractDinitrogen (N2) fixation was investigated at a pelagic station in the oligotrophic waters of the northern Gulf of Aqaba, Red Sea, between February 2016 and December 2018. In situ 15N2 and 13C incubations were used to evaluate photic and aphotic N2 fixation rates and diazotrophic contribution to water column productivity. N2 fixation rates were typically low (below detection to &lt;0.5 nmol N L−1 d−1). Maximal rates of 3.1 nmol L−1 d−1, measured at 100 m when conspicuous slicks of the cyanobacterium Trichodesmium appeared on the surface water. Amplicon sequencing of nifH demonstrated that non‐cyanobacterial diazotrophs, mostly α‐ and γ‐proteobacteria comprised the majority (82–100%) of amplicon sequence variants retrieved from photic and aphotic depths when low N2 fixation rates were measured, while amplicons representing cyanobacteria were nearly absent, but appeared in low abundance (~ 2%) when maximal rates were measured. During the stratified summer‐period, water‐column N2 fixation rates (10–75 μmol m−2 d−1) comprised ~ 1–40% of new production (NP). During the winter mixing period N2 fixation rates were considerably higher (11–242 μmol m−2 d−1) but made up only &lt;1% of NP. This is because, during this period, nitrate supplied to the photic zone by the vertical mixing becomes the major N source for NP. We conclude that on an annual average, diazotrophy plays a minor role in the NP of the Gulf. The major “new” nitrogen sources are cross‐thermocline turbulent diffusion of nitrate during summer stratification and vertical mixing during the fall–winter.

Nitrogen fixation in the western coastal Bay of Bengal: Controlling factors and contribution to primary production

N2 fixation rates were measured at 18 stations in the western coastal Bay of Bengal (BoB) to evaluate its contribution to the external source of nitrogen and its controlling factors. During the study period (fall monsoon), East India Coastal Currents (EICC) flows equatorward resulting in spreads of low saline water along the coast. This leads to stratification and inhibition of nutrient supply to the mixed layer through vertical mixing, resulting in the existence of nitrogen-poor and warm waters in the upper layer. Such conditions may trigger N2 fixation in the upper ocean. To examine such possibility, the N2 fixation rates were measured and found to vary between 0.4 and 14.8μmol N m−2 d−1 in the photic layer during the fall monsoon. A linear relationship was observed between photic zone integrated N2 fixation rates and phosphate suggesting a possible severe limitation of phosphate in the BoB. Low N2 fixation rates were observed at off Godavari, Krishna and Mahanadi River estuaries in the inshore than offshore regions associated with low phosphate and high suspended matter. This suggests that phosphate may be removed through adsorption on the suspended matter resulting in severe limitation of N2 fixation in the western BoB. Based on the available seasonal data, 0.01 to 0.94 Tg/y of N2 fixation occurs and contributes to <1% of the primary production in the BoB.

Coral-associated nitrogen fixation rates and diazotrophic diversity on a nutrient-replete equatorial reef

The role of diazotrophs in coral physiology and reef biogeochemistry remains poorly understood, in part because N2 fixation rates and diazotrophic community composition have only been jointly analyzed in the tissue of one tropical coral species. We performed field-based 15N2 tracer incubations during nutrient-replete conditions to measure diazotroph-derived nitrogen (DDN) assimilation into three species of scleractinian coral (Pocillopora acuta, Goniopora columna, Platygyra sinensis). Using multi-marker metabarcoding (16S rRNA, nifH, 18S rRNA), we analyzed DNA- and RNA-based communities in coral tissue and skeleton. Despite low N2 fixation rates, DDN assimilation supplied up to 6% of the holobiont’s N demand. Active coral-associated diazotrophs were chiefly Cluster I (aerobes or facultative anaerobes), suggesting that oxygen may control coral-associated diazotrophy. Highest N2 fixation rates were observed in the endolithic community (0.20 µg N cm−2 per day). While the diazotrophic community was similar between the tissue and skeleton, RNA:DNA ratios indicate potential differences in relative diazotrophic activity between these compartments. In Pocillopora, DDN was found in endolithic, host, and symbiont compartments, while diazotrophic nifH sequences were only observed in the endolithic layer, suggesting a possible DDN exchange between the endolithic community and the overlying coral tissue. Our findings demonstrate that coral-associated diazotrophy is significant, even in nutrient-rich waters, and suggest that endolithic microbes are major contributors to coral nitrogen cycling on reefs.

Temperate southern Australian coastal waters are characterised by surprisingly high rates of nitrogen fixation and diversity of diazotrophs.

Biological dinitrogen (N2) fixation is one mechanism by which specific microorganisms (diazotrophs) can ameliorate nitrogen (N) limitation. Historically, rates of N2 fixation were believed to be limited outside of the low nutrient tropical and subtropical open ocean; however, emerging evidence suggests that N2 fixation is also a significant process within temperate coastal waters. Using a combination of amplicon sequencing, targeting the nitrogenase reductase gene (nifH), quantitative nifH PCR, and 15N2 stable isotope tracer experiments, we investigated spatial patterns of diazotroph assemblage structure and N2 fixation rates within the temperate coastal waters of southern Australia during Austral autumn and summer. Relative to previous studies in open ocean environments, including tropical northern Australia, and tropical and temperate estuaries, our results indicate that high rates of N2 fixation (10–64 nmol L−1 d−1) can occur within the large inverse estuary Spencer Gulf, while comparatively low rates of N2 fixation (2 nmol L−1 d−1) were observed in the adjacent continental shelf waters. Across the dataset, low concentrations of NO3/NO2 were significantly correlated with the highest N2 fixation rates, suggesting that N2 fixation could be an important source of new N in the region as dissolved inorganic N concentrations are typically limiting. Overall, the underlying diazotrophic community was dominated by nifH sequences from Cluster 1 unicellular cyanobacteria of the UCYN-A clade, as well as non-cyanobacterial diazotrophs related to Pseudomonas stutzeri, and Cluster 3 sulfate-reducing deltaproteobacteria. Diazotroph community composition was significantly influenced by salinity and SiO4 concentrations, reflecting the transition from UCYN-A-dominated assemblages in the continental shelf waters, to Cluster 3-dominated assemblages in the hypersaline waters of the inverse estuary. Diverse, transitional diazotrophic communities, comprised of a mixture of UCYN-A and putative heterotrophic bacteria, were observed at the mouth and southern edge of Spencer Gulf, where the highest N2 fixation rates were observed. In contrast to observations in other environments, no seasonal patterns in N2 fixation rates and diazotroph community structure were apparent. Collectively, our findings are consistent with the emerging view that N2 fixation within temperate coastal waters is a previously overlooked dynamic and potentially important component of the marine N cycle.

N2 fixation, and the relative contribution of fixed N, in corals from Curaçao and Hawaii

Corals from Hawaii (Montipora capitata) and the Caribbean (brown and orange morphs of Montastraea cavernosa) have previously been shown to harbor symbiotic bacteria capable of fixing nitrogen (N2). Using a nitrogen tracer approach, we find that the rates of net photosynthesis and N2 fixation in M. capitata were significantly lower, while steady-state quantum yields of photosystem II (PSII) fluorescence (∆Fv/Fm′) were significantly higher, when compared to both color morphs of M. cavernosa where there was an inverse relationship between rates of photosynthesis and N2 fixation. However, the amount of fixed N contributing to Symbiodiniaceae N demand was consistent with their observed rates of N2 fixation. The lowest values occurred in M. capitata (0.034% ± 0.002 SE), and the brown morph of M. cavernosa had significantly lower values (1.081% ± 0.12 SE) compared to the orange morph (8.141% ± 0.36 SE). Additionally, for both the Symbiodiniaceae and microbial communities there were significant differences between species/color morphs where M. capitata was significantly different from both color morphs of M. cavernosa which were not significantly different from each other. An analysis of predicted metabolic activity, using PICRUSt2, also showed that the corals in this study were not predicted to be differentially enriched in genes involved in carbon metabolism, but genes involved in denitrification were predicted to be significantly enriched in both M. cavernosa orange and brown morphs. Genes involved in N2 fixation were, surprisingly, predicted to be enriched in M. cavernosa brown morphs. We suggest that the inhibition of nitrogenase by hyperoxia is one factor contributing to the low rates of N2 fixation in both M. capitata and M. cavernosa brown morphs.

H2 accumulation and N2 fixation variation by Ni limitation in Cyanothece

AbstractNi is an essential cofactor in NiFe‐uptake hydrogenase, an enzyme regulating H2 metabolism in diazotrophic cyanobacteria, the major H2 producers in the surface ocean globally. Here, we investigated the effect of Ni supply on H2 production and N2 fixation by using a model marine cyanobacterial diazotroph, Cyanothece. By mediating total dissolved Ni concentrations from 100 to 0.03 nmol L−1 in a trace metal‐defined culture medium, we demonstrated that Ni deficiency results in H2 accumulation, coupled with decreasing Ni quotas, growth rates, and occasionally relatively low N2 fixation rates. These results indicate that Ni deficiency limits the growth of the Cyanothece to some extent, considerably decreases H2 uptake by hydrogenase and leads to H2 accumulation and N2 fixation variation in the diazotroph. The findings show that Ni availability is a critical factor on controlling H2 production and N2 fixation in marine diazotrophic cyanobacteria. The information of Ni bioavailability for diazotrophic cyanobacteria is thus essential to evaluate the importance of Ni for H2 cycling and N2 fixation in oceanic surface waters.

Diazotrophy as the main driver of the oligotrophy gradient in the western tropical South Pacific Ocean: results from a one-dimensional biogeochemical–physical coupled model

Abstract. The Oligotrophy to UlTra-oligotrophy PACific Experiment (OUTPACE) cruise took place in the western tropical South Pacific (WTSP) during the austral summer (March–April 2015). The aim of the OUTPACE project was to investigate a longitudinal gradient of biological and biogeochemical features in the WTSP, and especially the role of N2 fixation in the C, N, and P cycles. Two contrasted regions were considered in this study: the Western Melanesian Archipelago (WMA), characterized by high N2 fixation rates, significant surface production and low dissolved inorganic phosphorus (DIP) concentrations, and the South Pacific Gyre (WGY), characterized by very low N2 fixation rates, surface production and high DIP concentrations. Since physical forcings and mixed layer dynamics in both regions were similar, it was considered that the gradient of oligotrophy observed in situ between the WMA and WGY was not explained by differences in physical processes, but rather by differences in biogeochemical processes. A one-dimensional physical–biogeochemical coupled model was used to investigate the role of N2 fixation in the WTSP by running two identical simulations, only differing by the presence (simWMA) or absence (simWGY) of diazotrophs. We showed that the nitracline and the phosphacline had to be, respectively, deeper and shallower than the mixed layer depth (MLD) to bring N-depleted and P-repleted waters to the surface during winter mixing, thereby creating favorable conditions for the development of diazotrophs. We also concluded that a preferential regeneration of the detrital phosphorus (P) matter was necessary to obtain this gap between the nitracline and phosphacline depths, as the nutricline depths significantly depend on the regeneration of organic matter in the water column. Moreover, the model enabled us to highlight the presence of seasonal variations in primary production and P availability in the upper surface waters in simWMA, where diazotrophs provided a new source of nitrogen (N) to the ecosystem, whereas no seasonal variations were obtained in simWGY, in the absence of diazotrophs. These main results emphasized the fact that surface production dynamics in the WTSP is based on a complex and sensitive system which depends on the one hand on physical processes (vertical mixing, sinking of detrital particles), and on the other hand on biogeochemical processes (N2 fixation, remineralization).

Distribution and rates of nitrogen fixation in the western tropical South Pacific Ocean constrained by nitrogen isotope budgets

Abstract. Constraining the rates and spatial distribution of dinitrogen (N2) fixation fluxes to the ocean informs our understanding of the environmental sensitivities of N2 fixation as well as the timescale over which the fluxes of nitrogen (N) to and from the ocean may respond to each other. Here we quantify rates of N2 fixation as well as its contribution to export production along a zonal transect in the western tropical South Pacific (WTSP) Ocean using N isotope (“δ15N”) budgets. Comparing measurements of water column nitrate + nitrite δ15N with the δ15N of sinking particulate N at a western, central, and eastern station, these δ15N budgets indicate high, modest, and low rates of N2 fixation at the respective stations. The results also imply that N2 fixation supports exceptionally high, i.e. ≥ 50 %, of export production at the western and central stations, which are also proximal to the largest iron sources. These geochemically based rates of N2 fixation are equal to or greater than those previously reported in the tropical North Atlantic, indicating that the WTSP Ocean has the capacity to support globally significant rates of N2 fixation, which may compensate for N removal in the oxygen-deficient zones of the eastern tropical Pacific.

Diversity and activity of nitrogen‐fixing communities across ocean basins

AbstractRecent observations of N2 fixation rates (NFR) and the presence of nitrogenase (nifH) genes from heterotrophic N2‐fixing (diazotrophic) prokaryotes in unusual habitats challenge the paradigm that pelagic marine N2 fixation is constrained to cyanobacteria in warm, oligotrophic, surface waters. Here, we compare NFR and diazotrophic diversity (assessed via high‐throughput nifH sequencing) from a region known to be dominated by cyanobacterial diazotrophs (the North Pacific Subtropical Gyre, NPSG) to two regions dominated by heterotrophic diazotrophs: the Eastern South Pacific (ESP, from the Chilean upwelling system to the subtropical gyre) and the Pacific Northwest coastal upwelling system (PNW). We observed distinct biogeographical patterns among the three regions. Diazotrophic community structure differed strongly between the NPSG, dominated by cyanobacterium UCYN‐A, and the ESP, dominated by heterotrophic nifH group 1J/1K, yet surface NFR were similar in magnitude (up to 5.1 nmol N L−1 d−1). However, while diverse, predominantly heterotrophic nifH genes were recovered from the PNW and the mesopelagic of the NPSG, NFR were undetectable in both of these environments (although glucose amendments stimulated low rates in the deep NPSG). Our work suggests that while diazotrophs may be nearly omnipresent in marine waters, the activity of this functional group is regionally restricted. Further, we show that the detection limits of the 15N2 fixation assay suggest that many of the low NFR reported for the mesopelagic (often &lt; 0.1 nmol N L−1 d−1 in the literature) are not indicative of active diazotrophy, highlighting the challenges of assessing the ecosystem significance of heterotrophic diazotrophs.

Evaluating the balance between vertical diffusive nitrate supply and nitrogen fixation with reference to nitrate uptake in the eastern subtropical North Atlantic Ocean

The balance between N2 fixation and diffusive NO3? supply is a key determinant for assessing the importance of both processes for new production in subtropical waters. Here we report observations of integrated N2 fixation rates from the eastern subtropical North Atlantic Ocean with coincident estimates of diffusive NO3? supply. We find the average rate of N2 fixation is equivalent to 62% of the diffusive NO3? supply, though N2 fixation could exceed the diffusive flux at individual stations. Turbulent diffusivity measurements across the nitracline indicate a mean diffusivity of 0.077 cm2 s?1. If approximations for methodological underestimates in the dominant N2 fixation technique are considered, the magnitude of N2 fixation is shown to represent 100% of the NO3? flux on average, and can be almost threefold higher at individual stations. As the study site is characterized by low rates of N2 fixation compared to other sectors of the North Atlantic this confirms N2 fixation as a major source term across the subtropical North Atlantic. The seasonal context of our observations suggests environmental factors underlie the in situ variability in observed N2 fixation rates, and may well explain lower previous assessments of the importance of N2 fixation relative to diffusive NO3? supply in this region. The diffusive NO3? supply provides <20% of measurable NO3? uptake with the remainder supplied via other mechanisms, most notably nitrification. The mean integrated rate of N2 fixation equates to just 8% of the NO3? consumed on a daily basis by the phytoplankton community.

Uncoupling between dinitrogen fixation and primary productivity in the eastern Mediterranean Sea

In the nitrogen (N)‐impoverished photic zones of many oceanic regions, prokaryotic organisms fixing atmospheric dinitrogen (N2; diazotrophs) supply an essential source of new nitrogen and fuel primary production. We measured dinitrogen fixation and primary productivity (PP) during the thermally stratified summer period in different water regimes of the oligotrophic eastern Mediterranean Sea, including the Cyprus Eddy and the Rhodes Gyre. Low N2 fixation rates were measured (0.8–3.2 µmol N m−2 d−1) excluding 10‐fold higher rates in the Rhodes Gyre and Cyprus Eddy (~20 µmol N m−2 d−1). The corresponding PP increased from east to west (200–2500 µmol C m−2 d−1), with relatively higher productivity recorded in the Rhodes Gyre and Cyprus Eddy (2150 and 2300 µmol C m−2 d−1, respectively). These measurements demonstrate that N2 fixation in the photic zone of the eastern Mediterranean Sea contributes only negligibly by direct inputs to PP (i.e., cyanobacterial diazotrophs) and is in fact uncoupled from PP. By contrast, N2 fixation is significantly coupled to bacterial productivity and to net heterotrophic areas, suggesting that heterotrophic N2 fixation may in fact be significant in this ultraoligotrophic system. This is further substantiated by the high N2 fixation rates we measured from aphotic depths and by the results of phylogenetic analysis in other studies showing an abundance of heterotrophic diazotrophs.

First basin‐wide experimental results on N2 fixation in the open Mediterranean Sea

The Mediterranean Sea presents several biogeochemical anomalies compared to the global ocean. An unbalanced N budget, high nitrate/phosphate ratios in subsurface waters and low 15N/14N ratios in particulate and dissolved nitrogen suggest a significant occurrence of N2 fixation. This study presents, for the first time, a basin‐wide overview of direct measurements of N2 fixation, with values in the North Atlantic for comparison, during late spring 2007. Very low N2 fixation rates (0.052 ± 0.031 nmols N l−1d−1) were observed in all sub‐regions of the Mediterranean, in contrast to the higher values measured in the North Atlantic (0.300 ± 0.115 nmols N l−1d−1). Higher phosphorus (inorganic or organic) concentrations were not associated with higher N2 fixation rates. Low 15N/14N ratios in particulate organic nitrogen (from −2.10 to +4.11‰), associated with low N2 fixation rates, suggest that other N sources, such as atmospheric inputs, fuel the Mediterranean ecosystem.

Nitrogen fixation and biomass productivity of indigenous legumes for fertility restoration of abandoned soils in smallholder farming systems

Most legume-based soil fertility technologies often fail to make the desired impact on nutrient-depleted soils partly due to low N2-fixation rates and poor biomass productivity. A study was conducted in the 2004/05 and 2005/06 rainfall seasons to evaluate biomass productivity and N2-fixation of indigenous legumes on nutrient-depleted fields under low (450- 650 mm yr−1) to high (> 800 mm yr−1) rainfall areas of Zimbabwe. Legume species, mostly of Crotalaria, Indigofera and Tephrosia genera, were sown in mixtures on disturbed soil. Indigenous legume fallows (indifallows) produced 2–5 t biomass ha−1 under low and 5–15 t biomass ha−1 under high rainfall. They significantly (P<0.05) out-yielded natural fallows by 84% and a sunnhemp (Crotalaria juncea) green manure fallow by 32% over a growth period of six months. Cumulatively, indifallows produced more biomass (~29 t ha−1) than natural fallows (~ 8 t ha−1) over two seasons. Indigenous legumes derived 61–90% of their N from the atmosphere with amounts fixed ranging from 2–57 kg N ha−1 under medium rainfall conditions to 1–173 kg N ha−1 under high rainfall. Application of P increased indifallow biomass productivity by 15% under low rainfall, and N2-fixation by 32% and 18% under low and high rainfall, respectively. These results demonstrated that indigenous legumes generate sufficiently high biomass and fix nitrogen on nutrient-depleted fields where most conventional green manure and grain legume cultivars often fail to establish.

Nitrogen fixation by unicellular diazotrophic cyanobacteria in the temperate oligotrophic North Pacific Ocean

N2 fixation has been understudied in marine environments outside of the subtropical and tropical oceans and where water temperatures are typically below 20‐25°C. We identified nifH phylotypes and measured N2 fixation rates under ambient conditions (maximum of 19°C) in water collected 750 km off the coast of California in oligotrophic waters of the North Pacific Ocean (34°N, 129°W). Near‐surface N2 fixation rates averaged 0.25 ± 0.05 nmol N L−1 d−1 for 24 incubation bottles. Despite low ambient concentrations of iron (&lt;0.1 nmol L−1) and phosphorus (&lt;0.3 µmol L−1), N2 fixation rates were unaffected by iron and phosphorus amendments. Using reverse transcription‐quantitative polymerase chain reaction (RT‐QPCR) methodology, we estimated transcript abundance and patterns of expression for several unicellular diazotrophs, including the group A phylotype, which showed the highest daily mRNA abundances. The N2‐fixing assemblage extended to 60‐80 m depth, well below the seasonal thermocline (40 m). The calculated areal N2 fixation rate (15 µmol N m−2 d−1) was small compared with estimates from other regions of the Pacific; however, the estimated fixation rate was similar to other published results, suggesting that processes other than cellular growth rate may determine the abundance of unicellular diazotrophs. Despite the low N2 fixation rates, the new nitrogen added to the euphotic zone by N2 fixation could account for at least 10% of new production during the study period.

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover.

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.

RAPID N2FIXATION IN PINES? RESULTS OF A MAINE FIELD STUDY

A recent study of the nitrogen budgets of pitch pines (Pinus rigida Mill.) and red pines (P. resinosa Ait.) growing in model ecosystems in New Hampshire (USA) reported very high nitrogen gains. Thus N2 fixation associated with the rhizosphere of pines may contribute to their success in nitrogen-limited ecosystems. We used the acetylene-reduction assay with carbon monoxide controls to determine whether high rates of N2 fixation are associated with pine forests in Maine. We measured nitrogenase activity in cores of the forest floor and the mineral soil beneath for five pitch pine, seven red pine, and six eastern white pine (P. strobus L.) stands. The stands represented 8 of the 15 biophysical regions of Maine (USA) and included a variety of age classes. We found only very low rates of nitrogenase activity, which varied seasonally with the highest values during the warmer months. The highest mean nitrogenase activity observed was 449 nmol C2H4·m−2·h−1. We estimated that <6 mg N/m2 was fixed annually by non-symbiotic diazotrophs in the soil horizons sampled. These rates are much smaller than atmospheric N inputs in Maine (300–450 mg N·m−2·yr−1) and consequently have little impact on the N budgets of the pines. Based on our results and the literature, it is likely that only low rates of N2 fixation are associated with pines.

Untraditional N2-fixing bacteria as biofertilizers for wheat and barley

The screening of 27 isolates grown on nitrogen-free medium for nitrogen-fixing ability resulted in the isolation of five organisms belonging toBacillaceae, Enterobacteriaceae andPseudomonadaceae. Estimates of N2-fixation efficiencies of these isolates indicated that they may be responsible for low rates of N2-fixation in soil. The possible association of these isolates as well as ofAzotobacter andAzospirillum with wheat and barley was investigated in a greenhouse experiment. The highest values of nitrogenase activity on plant root were recorded in treatments inoculated with composite inocula of the isolated N2-fixers, particularly whenAzotobacter and/orAzospirillum were added in combination. Inoculation with single inoculum of each of the N2-fixing isolates had no significant influence on plant growth, except withPseudomonas andBacillus for wheat and barley, respectively. Highly significant increases in growth of both plants were recorded in all cases of multistrain inoculation.