Biological Dinitrogen Fixation Research Articles

Structural comparison of (hyper-)thermophilic nitrogenase reductases from three marine Methanococcales.

The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N2) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N2-machinery from these ancient archaea.

Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

Improving semi-arid agroecosystem services with cover crop mixes.

Winter wheat (Triticum aestivum, L.) production in the semi-arid US Northern High Plains (NHP) is challenged by frequent droughts and water-limited, low fertility soils. Composted cattle manure (compost) and cover crops (CC) are known to provide agroecosystem services such as improved soil health, and in the CC case, increased plant diversity, and competition with weedy species. The main concern of planting CC in winter wheat fallow rotation in regions that are more productive than the NHP, however, is the soil moisture depletion. It is unknown however, whether addition of CC to compost-amended soils in the NHP will improve soil properties and agroecosystem health without compromising already low soil water content. The main objective of this study was to assess the effects of four CC treatments amended with compost (45 Mg ha-1) or inorganic fertilizer (IF) (.09 Mg ha-1 mono-ammonium phosphate, 11-52-0 and 1.2 Mg ha-1ammonium sulfate, 21-0-0) on the presence of weeds, soil and plant total carbon (C), nitrogen (N), and biological dinitrogen (N2) fixation (BNF). Mycorrhizal Mix (MM), Nitrogen Fixer Mix (NF), Soil Building Mix (SB), a monoculture of phacelia (Phacelia tanacetifolia Benth L.) (PH), and a no CC control (no CC) were grown in native soil kept at 7% soil moisture in a greenhouse for a period of nine weeks. When amended with compost, MM was the most beneficial (48 g m-2 BNF and 1.7% soil C increase). SB had the highest germination, aboveground biomass, and decreased weed biomass by 60%. It also demonstrated the second highest amount of BNF (40 g m-2) and soil C increase by 1.5%. On contrary, IF hindered BNF by almost 70% in all legume-containing CC treatments and reduced soil C by 15%.

Phenological and Morphological Response of Chickpea (Cicer arietinum L.) Rhizobia and Azotobacter Inoculation

Excessive use of nitrogen has become a threat to human health and the environment due to high concentrations of nitrate and nitrite accumulating in surface and ground waters. Biological dinitrogen fixation (N2) is a very important natural process in world agriculture. Rhizobia is a common name for a certain Gram-negative group of Alphaproteobacteria and Betaproteobacteria that can form nodules on the root and fix nitrogen in symbiosis with legumes as their host plants. Azotobacter spp. is a free-living microorganism that has the ability to fix atmospheric nitrogen into the soil. Field trials were carried out of Eskişehir Osmangazi University Field Crops Department experiment areas during the production season of 2017 and 2019. The study was evaluated the effects on morphological and phenologocial characters of two N doses (0 and 25 kg ha-1 N), four bacteria inoculations (control, Rhizobia, Azotobacter, Rhizobia + Azotobacter) on chickpea cultivars (Azkan, Akca, Cakir, Isik). Experiment arranged in split split plot experimental design with three replications. Nitrogen application positively affected number of days to emergence, number of branches, branch diameter and grain yield. Phenological and morphological properties affected by climate conditions. The high temperature caused the number of days to emergence, number of days to flowering and number of days to maturity to be earlier in the second year. Plant height, first pod height and grain yield were higher first year than second year due to high precipitation. While the response of the cultivars was different in terms of phenological properties, Azkan cultivar gave the best results in terms of morphological properties. Rhizobia + Azotobacter inoculation gave the best results in terms of phenological and morphological characteristics except for number of days to maturity. Rhizobia + Azotobacter inoculation can lead to additional income generation of the farming community in Turkey. The use of biofertilizers may reduce the application of chemical fertilizers and we get healthy, pollution-free production for a better future for our increasing populations.

Nitrogenases in Oxygen Minimum Zone Waters

Biological dinitrogen (N2) fixation is the pathway making the large pool of atmospheric N2 available to marine life. Besides direct rate measurements, a common approach to explore the potential for N2 fixation in the ocean is a screening-based targeting the key functional marker gene nifH, coding for a subunit of the nitrogenase reductase. As novel sequencing techniques improved, our understanding of the diversity of marine N2 fixers grew exponentially. However, one aspect of N2 fixation in the ocean is often underexplored, which are the two alternative types of the key enzyme of N2 fixation, the nitrogenase. Altogether there are three isoenzymes, the most common Mo-Fe nitrogenase Nif, the Fe-Fe nitrogenase Anf, and the V-Fe nitrogenase Vnf, which differ regarding their genetic organization, as well as their metal co-enzymes. While Mo is only available in the presence of at least traces of oxygen (O2), V and Fe are available if O2 is absent. Therefore, low O2 and anoxic ocean environments could be an ideal place to explore the diversity of the different isotypes of the nitrogenases. Most phylogenetic studies, however, were only based on the functional marker gene nifH, encoding for a subunit of the Nif nitrogenase, and thus limited in representing the diversity of alternative nitrogenases. Here, we screened metagenomes and -transcriptomes from O2 minimum zones off Peru, from the Bay of Bengal, and the anoxic Saanich Inlet to explore the diversity of genes involved in N2 fixation. We identified genes related to all three nitrogenases, and a generally increased diversity as compared to our previous nifH based on studies from OMZ waters. While we could not confirm gene expression of alternative nitrogenases from our transcriptomic, we detected diazotrophs harboring the genetic potential for alternative nitrogenases. We suggest that alternative nitrogenases may not be used under conditions present in those waters, however, depending on trace metal availability they may become active under future ocean deoxygenation.

The Angola Gyre is a hotspot of dinitrogen fixation in the South Atlantic Ocean

Biological dinitrogen fixation is the major source of new nitrogen to marine systems and thus essential to the ocean’s biological pump. Constraining the distribution and global rate of dinitrogen fixation has proven challenging owing largely to uncertainty surrounding the controls thereon. Existing South Atlantic dinitrogen fixation rate estimates vary five-fold, with models attributing most dinitrogen fixation to the western basin. From hydrographic properties and nitrate isotope ratios, we show that the Angola Gyre in the eastern tropical South Atlantic supports the fixation of 1.4–5.4 Tg N.a−1, 28-108% of the existing (highly uncertain) estimates for the basin. Our observations contradict model diagnoses, revealing a substantial input of newly-fixed nitrogen to the tropical eastern basin and no dinitrogen fixation west of 7.5˚W. We propose that dinitrogen fixation in the South Atlantic occurs in hotspots controlled by the overlapping biogeography of excess phosphorus relative to nitrogen and bioavailable iron from margin sediments. Similar conditions may promote dinitrogen fixation in analogous ocean regions. Our analysis suggests that local iron availability causes the phosphorus-driven coupling of oceanic dinitrogen fixation to nitrogen loss to vary on a regional basis.

High Diazotrophic Diversity but Low N2 Fixation Activity in the Northern Benguela Upwelling System Confirming the Enigma of Nitrogen Fixation in Oxygen Minimum Zone Waters

Oxygen minimum zones (OMZs) have been suggested as a suitable niche for the oxygen-sensitive process of biological fixation of dinitrogen (N2) gas. However, most N2 fixation rates reported from such waters are low. This low N2 fixation activity has been proposed to result from the unusual community of N2 fixers, in which cyanobacteria were typically underrepresented. The Northern Benguela Upwelling System (North BUS) is part of one of the most productive marine ecosystems and hosts a well-developed OMZ. Although previous observations indicated low to absent N2 fixation rates, the community composition of diazotrophs needed to understand the North BUS has not been described. Here, we present a first detailed analysis of the diazotrophic diversity in the North BUS OMZ and the Angola tropical zone (ATZ), based on genetic data and isotope speciation. Consistent with a previous study, we detected a slight N deficit in the OMZ, but isotope data did not indicate any active or past N2 fixation. The diazotroph community in the North BUS was dominated by non-cyanobacterial microbes clustering with members of gamma-proteobacteria, as is typical for other OMZ regions. However, we found a strikingly high diversity of Cluster III diazotrophs not yet described in other OMZs. In contrast to previous observations, we could also identify cyanobacteria of the clades Trichodesmium sp., UCYN-A and Cyanothece sp., in surface waters connected to or above the OMZ, which were potentially active as shown by the presence of genes and transcripts of the key functional marker gene for N2 fixation, nifH. While the detection of diazotrophs and the absence of active N2 fixation (based on isotopic speciation) are consistent with other OMZ observations, the detected regional variation in the diversity and presence of cyanobacteria indicate that we still are far from understanding the role of diazotrophs in OMZs, which, however, is relevant for understanding the N cycle in OMZ waters, as well for predicting the future development of OMZ biogeochemistry in a changing ocean.

Planktonic Aggregates as Hotspots for Heterotrophic Diazotrophy: The Plot Thickens.

Biological dinitrogen (N2) fixation is performed solely by specialized bacteria and archaea termed diazotrophs, introducing new reactive nitrogen into aquatic environments. Conventionally, phototrophic cyanobacteria are considered the major diazotrophs in aquatic environments. However, accumulating evidence indicates that diverse non-cyanobacterial diazotrophs (NCDs) inhabit a wide range of aquatic ecosystems, including temperate and polar latitudes, coastal environments and the deep ocean. NCDs are thus suspected to impact global nitrogen cycling decisively, yet their ecological and quantitative importance remain unknown. Here we review recent molecular and biogeochemical evidence demonstrating that pelagic NCDs inhabit and thrive especially on aggregates in diverse aquatic ecosystems. Aggregates are characterized by reduced-oxygen microzones, high C:N ratio (above Redfield) and high availability of labile carbon as compared to the ambient water. We argue that planktonic aggregates are important loci for energetically-expensive N2 fixation by NCDs and propose a conceptual framework for aggregate-associated N2 fixation. Future studies on aggregate-associated diazotrophy, using novel methodological approaches, are encouraged to address the ecological relevance of NCDs for nitrogen cycling in aquatic environments.

Global Grassland Diazotrophic Communities Are Structured by Combined Abiotic, Biotic, and Spatial Distance Factors but Resilient to Fertilization

Grassland ecosystems cover around 37% of the ice-free land surface on Earth and have critical socioeconomic importance globally. As in many terrestrial ecosystems, biological dinitrogen (N2) fixation represents an essential natural source of nitrogen (N). The ability to fix atmospheric N2 is limited to diazotrophs, a diverse guild of bacteria and archaea. To elucidate the abiotic (climatic, edaphic), biotic (vegetation), and spatial factors that govern diazotrophic community composition in global grassland soils, amplicon sequencing of the dinitrogenase reductase gene—nifH—was performed on samples from a replicated standardized nutrient [N, phosphorus (P)] addition experiment in 23 grassland sites spanning four continents. Sites harbored distinct and diverse diazotrophic communities, with most of reads assigned to diazotrophic taxa within the Alphaproteobacteria (e.g., Rhizobiales), Cyanobacteria (e.g., Nostocales), and Deltaproteobacteria (e.g., Desulforomonadales) groups. Likely because of the wide range of climatic and edaphic conditions and spatial distance among sampling sites, only a few of the taxa were present at all sites. The best model describing the variation among soil diazotrophic communities at the OTU level combined climate seasonality (temperature in the wettest quarter and precipitation in the warmest quarter) with edaphic (C:N ratio, soil texture) and vegetation factors (various perennial plant covers). Additionally, spatial variables (geographic distance) correlated with diazotrophic community variation, suggesting an interplay of environmental variables and spatial distance. The diazotrophic communities appeared to be resilient to elevated nutrient levels, as 2–4 years of chronic N and P additions had little effect on the community composition. However, it remains to be seen, whether changes in the community composition occur after exposure to long-term, chronic fertilization regimes.

Straw Mulching and Nitrogen Fertilization Affect Diazotroph Communities in Wheat Rhizosphere

Diazotrophs that carry out the biological fixation of atmospheric dinitrogen (N2) replenish biologically available nitrogen (N) in soil and are influenced by the input of inorganic and organic substrates. To date, little is known about the effects of combined organic substrate addition and N fertilization on the diazotroph community composition and structure in purple soils. We investigated the effects of N fertilization and straw mulching on diazotroph communities by quantifying and sequencing the nifH gene in wheat rhizosphere. The abundance and richness of diazotrophs were greater the higher the fertilization level in the mulched treatments, whereas in the nonmulched treatments (NSMs), richness was lowest with the highest N fertilization level. The abundance and α-diversity of diazotrophs correlated with most of the soil properties but not with pH. At the genus level, the relative abundances of Azospirillum, Bacillus, and Geobacter were higher in the NSMs and those of Pseudacidovorax, Skermanella, Azospira, Paraburkholderia, Azotobacter, Desulfovibrio, Klebsiella, and Pelomonas in the mulched treatments. The differences in community composition between the mulched and the NSMs were associated with differences in soil temperature and soil organic carbon and available potassium contents and C:N ratio. Overall, straw mulching and N fertilization were associated with changes in diazotroph community composition and higher abundance of nifH gene in alkaline purple soils.

Evaluation of argon-induced hydrogen production as a method to measure nitrogen fixation by cyanobacteria.

The production of dihydrogen (H2 ) is an enigmatic yet obligate component of biological dinitrogen (N2 ) fixation. This study investigates the effect on H2 production by N2 fixing cyanobacteria when they are exposed to either air or a gas mixture consisting of argon, oxygen, and carbon dioxide (Ar:O2 :CO2 ). In the absence of N2 , nitrogenase diverts the flow of electrons to the production of H2 , which becomes a measure of Total Nitrogenase Activity (TNA). This method of argon-induced hydrogen production (AIHP) is much less commonly used to infer rates of N2 fixation than the acetylene reduction (AR) assay. We provide here a full evaluation of the AIHP method and demonstrate its ability to achieve high-resolution measurements of TNA in a gas exchange flow-through system. Complete diel profiles of H2 production were obtained for N2 fixing cyanobacteria despite the absence of N2 that broadly reproduced the temporal patterns observed by the AR assay. Comparison of H2 production under air versus Ar:O2 :CO2 revealed the efficiency of electron usage during N2 fixation and place these findings in the broader context of cell metabolism. Ultimately, AIHP is demonstrated to be a viable alternative to the AR assay with several additional merits that provide an insight into cell physiology and promise for successful field application.

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.

Bridging the Spatiotemporal Gap in Diazotroph Activity and Diversity With High-Resolution Measurements

The biological fixation of dinitrogen (N 2) by marine microbes called diazotrophs sustains ∼50% new production in the ocean, boosting CO 2 absorption by photoautotrophs and thus contributing to the mitigation of climate change. New environmental conditions sustaining N 2 fixation have been revealed in recent years, enabling more accurate forecasting of future nitrogen inputs and localized hot spots. However, at present the paucity and biased geographical coverage of N 2 fixation and diazotroph diversity measurements impede attempts to reconcile global nitrogen budgets with observed rates. Most studies have been conducted at disparate spatiotemporal scales, including: (i) discrete and short duration measurements in small seawater volumes isolated from the environment, and (ii) spatial extrapolations and global models of diazotrophy projected over decades to centuries. We argue that this knowledge gap lies at the fine scales: dynamic seawater structures < 200 km wide and < 2 months lifetime. However, the spatiotemporal resolution of conventional oceanographic cruises, with stations separated by tens to hundreds of kilometers, is too poor to resolve fine scale processes. Bridging this gap requires leveraging high spatiotemporal resolution measurements. Here we present and discuss the advantages and disadvantages of contemporary methods and equipment able to provide high-resolution measurements at sea. We also provide insights into high-resolution sampling approaches to be developed in the near future. Increasing the spatiotemporal resolution of diazotroph activity and diversity will provide more realistic quantifications of nitrogen fluxes in the dynamic ocean.

Dinitrogen fixation rates in the Bay of Bengal during summer monsoon

Biological dinitrogen (N2) fixation exerts an important control on oceanic primary production by providing bioavailable form of nitrogen (such as ammonium) to photosynthetic microorganisms. N2 fixation is dominant in nutrient poor and warm surface waters. The Bay of Bengal is one such region where no measurements of phototrophic N2 fixation rates exist. The surface water of the Bay of Bengal is generally nitrate-poor and warm due to prevailing stratification and thus, could favour N2 fixation. We commenced the first N2 fixation study in the photic zone of the Bay of Bengal using 15N2 gas tracer incubation experiment during summer monsoon 2018. We collected seawater samples from four depths (covering the mixed layer depth of up to 75 m) at eight stations. N2 fixation rates varied from 4 to 75 μmol N m−2 d−1. The contribution of N2 fixation to primary production was negligible (<1%). However, the upper bound of observed N2 fixation rates is higher than the rates measured in other oceanic regimes, such as the Eastern Tropical South Pacific, the Tropical Northwest Atlantic, and the Equatorial and Southern Indian Ocean.

No nitrogen fixation in the Bay of Bengal?

Abstract. The Bay of Bengal (BoB) has long stood as a biogeochemical enigma, with subsurface waters containing extremely low, but persistent, concentrations of oxygen in the nanomolar range which – for some, yet unconstrained, reason – are prevented from becoming anoxic. One reason for this may be the low productivity of the BoB waters due to nutrient limitation and the resulting lack of respiration of organic material at intermediate waters. Thus, the parameters determining primary production are key in understanding what prevents the BoB from developing anoxia. Primary productivity in the sunlit surface layers of tropical oceans is mostly limited by the supply of reactive nitrogen through upwelling, riverine flux, atmospheric deposition, and biological dinitrogen (N2) fixation. In the BoB, a stable stratification limits nutrient supply via upwelling in the open waters, and riverine or atmospheric fluxes have been shown to support only less than one-quarter of the nitrogen for primary production. This leaves a large uncertainty for most of the BoB's nitrogen input, suggesting a potential role of N2 fixation in those waters. Here, we present a survey of N2 fixation and carbon fixation in the BoB during the winter monsoon season. We detected a community of N2 fixers comparable to other oxygen minimum zone (OMZ) regions, with only a few cyanobacterial clades and a broad diversity of non-phototrophic N2 fixers present throughout the water column (samples collected between 10 and 560 m water depth). While similar communities of N2 fixers were shown to actively fix N2 in other OMZs, N2 fixation rates were below the detection limit in our samples covering the water column between the deep chlorophyll maximum and the OMZ. Consistent with this, no N2 fixation signal was visible in δ15N signatures. We suggest that the absence of N2 fixation may be a consequence of a micronutrient limitation or of an O2 sensitivity of the OMZ diazotrophs in the BoB. Exploring how the onset of N2 fixation by cyanobacteria compared to non-phototrophic N2 fixers would impact on OMZ O2 concentrations, a simple model exercise was carried out. We observed that both photic-zone-based and OMZ-based N2 fixation are very sensitive to even minimal changes in water column stratification, with stronger mixing increasing organic matter production and export, which can exhaust remaining O2 traces in the BoB.

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.

Diazotrophic microbial community and abundance in acidic subtropical natural and re-vegetated forest soils revealed by high-throughput sequencing of nifH gene

Biological nitrogen fixation (BNF) is an important natural biochemical process converting the inert dinitrogen gas (N2) in the atmosphere to ammonia (NH3) in the N cycle. In this study, the nifH gene was chosen to detect the diazotrophic microorganisms with high-throughput sequencing from five acidic forest soils, including three natural forests and two re-vegetated forests. Soil samples were taken in two seasons (summer and winter) at two depth layers (surface and lower depths). A dataset of 179,600 reads obtained from 20 samples were analyzed to provide the microbial community structure, diversity, abundance, and relationship with physiochemical parameters. Both archaea and bacteria were detected in these samples and diazotrophic bacteria were the dominant members contributing to the biological dinitrogen fixation in the acidic forest soils. Cyanobacteria, Firmicutes, Proteobacteria, Spirocheates, and Verrucomicrobia were observed, especially the Proteobacteria as the most abundant phylum. The core genera were Bradyrhizobium and Methylobacterium from α-Proteobacteia, and Desulfovibrio from δ-Proteobacteia in the phylum of Proteobacteia of these samples. The diversity indices and the gene abundances of all samples were higher in the surface layer than the lower layer. Diversity was apparently higher in re-vegetated forests than the natural forests. Significant positive correlation to the organic matter and nitrogen-related parameters was observed, but there was no significant seasonal variation on the community structure and diversity in these samples between the summer and winter. The application of high-throughput sequencing method provides a better understanding and more comprehensive information of diazotrophs in acidic forest soils than conventional and PCR-based ones.

Environmental and nutrient controls of marine nitrogen fixation

Biological dinitrogen (N2) fixation by diazotrophic cyanobacteria has great biogeochemical implications in nitrogen (N) cycling in the ocean as this process represents the major source of new N input to the oceans, thereby controlling the marine primary productivity. Numerous factors can affect the extent of N2 fixation. A better understanding of the major environmental and nutrient factors governing this process is highly required. Iron (Fe) and/or phosphorus (P) are thought to be limiting factors in most oceanic regions. Special attention has been given in the present study to evaluate the effects of mineral dust deposition which is believed to stimulate N2 fixation as it increases the availability of both Fe and P. Through three laboratory bioassays (+Fe, +P, +Dust) via incubation experiments performed on Trichodesmium IMS101, we found that each addition of Fe, P or desert dust could stimulate the growth and N2 fixation of Trichodesmium IMS101. Several adaptive nutrient utilization strategies were observed, such as a Fe luxury uptake mechanism, a P-sparing effect and colony formation. In addition, during a field study using natural phytoplankton assemblages from the temperate Northeast Atlantic Ocean the critical role of dissolved Fe (DFe) was again highlighted by the remarkably enhanced N2 fixation rate observed after the addition of DFe under low temperature and P-depleted conditions. At the time of our study no Trichodesmium filaments were found in the studied region, the diazotrophic community was dominated by unicellular cyanobacteria symbiont (prymnesiophyte-UCYN-A1) and heterotrophic diazotrophs, therefore demonstrating that DFe could as well be the ultimate factor limiting N2 fixation of these smaller diazotrophs. Recently, the effects of ongoing climate change (ocean warming and acidification) on N2 fixation drew much attention, but various studies led to controversial conclusions. Semi-continuous dilution growth experiments were conducted on Trichodesmium IMS101 under present-day and future high pCO2 (400 and 800 µatm, respectively) and warming seawater (24 and 28 °C) conditions. The results indicate that higher pCO2 and therefore ocean acidification may be beneficial for Trichodesmium growth and N2 fixation. However, our observations suggest that Fe or P limitation in oligotrophic seawaters may offset the stimulation induced on Trichodesmium IMS101 resulting from ocean acidification. In contrast, ocean warming may not play an important role in Trichodesmium growth and N2 fixation with a 4 °C increase from 24 °C to 28 °C. Nevertheless, ocean warming is predicted to cause a shift in the geographical distribution of Trichodesmium species toward higher latitudes, extending its niche to subtropical ocean regions and potentially reducing its coverage in tropical ocean basins.

Effectiveness of eco-friendly arbuscular mycorrhizal fungi biofertilizer and bacterial feather hydrolysate in promoting growth of Vicia faba in sandy soil

Abstract The current study purposed to improve vegetative growth, nodules formation and biological dinitrogen fixation of Vicia faba L. (faba bean) grown in sandy soil by inoculation with bacterial feather hydrolysate fertilizer (BFH) and arbuscular mycorrhizal fungi (AMF). A potent feather-degrading bacterium was isolated from chicken feathers and molecular identified as Bacillus licheniformis ASU (Accession number KF446641 ). Two different native AMF were recovered from the sandy soil and identified as Acaulospora bireticulata and Glomus caesaris. Bacillus licheniformis has the capability to degrade the chicken feathers efficiently. Furthermore, it can solubilize tricalcium phosphate and produce ammonia and IAA (indole acetic acid). Faba bean seeds were grown in pot experiments containing sandy soil inoculated with AMF or BFH individually or in combination. The application of AMF and BFH in combination significantly increased nodule formation and N2-fixation. The total dry biomass of plants co-inoculated with AMF and BFH was increased significantly compared with non-inoculated plants. So, the combination with AMF and BFH has the potential to be developed as a useful biofertilizer in future. This paper is expected to afford a competitive economic outcome for sustainable cropping systems.

Magnesium promotes root nodulation through facilitation of carbohydrate allocation in soybean.

Magnesium (Mg) is an essential element for the growth of both plants and bacteria. Low availability of Mg in agriculture can limit crop productivity and quality. In addition to direct effects on plant growth, limited Mg supply may also impact biological dinitrogen (N2 ) fixation in nodules formed from symbiotic interactions between legumes and rhizobial bacteria. To date, the physiological mechanisms involved in Mg-dependent nodulation remains largely unknown. The objectives of this work were to assess how Mg supply affects nodule growth and development in symbiotic systems, and to test if any observed changes in nodule and soybean are correlated with Mg supply. Here, we found that external Mg supply enhanced nodule growth under nitrogen (N) limited conditions, and subsequently improved N2 fixation and soybean growth. Mg supply altered neither nodule structure nor Mg homeostasis, but remarkably promoted nodule enlargement, resulting in an increase in the number of big nodules. In addition, high Mg supply decreased starch and sucrose accumulation in leaves, and increased their concentrations in roots, which consequently enhanced carbohydrate import into the rhizobia infection zone of nodules. In this study, Mg was shown to promote nodule growth in soybean. This Mg-promoted nodule growth is derived from Mg-facilitated alteration of carbohydrate partitioning and transport into nodules.