Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

Bryophyte-Cyanobacteria Associations during Primary Succession in Recently Deglaciated Areas of Tierra del Fuego (Chile)

Bryophyte establishment represents a positive feedback process that enhances soil development in newly exposed terrain. Further, biological nitrogen (N) fixation by cyanobacteria in association with mosses can be an important supply of N to terrestrial ecosystems, however the role of these associations during post-glacial primary succession is not yet fully understood. Here, we analyzed chronosequences in front of two receding glaciers with contrasting climatic conditions (wetter vs drier) at Cordillera Darwin (Tierra del Fuego) and found that most mosses had the capacity to support an epiphytic flora of cyanobacteria and exhibited high rates of N₂ fixation. Pioneer moss-cyanobacteria associations showed the highest N₂ fixation rates (4.60 and 4.96 µg N g⁻¹ bryo. d⁻¹) very early after glacier retreat (4 and 7 years) which may help accelerate soil development under wetter conditions. In drier climate, N₂ fixation on bryophyte-cyanobacteria associations was also high (0.94 and 1.42 µg N g⁻¹ bryo. d⁻¹) but peaked at intermediate-aged sites (26 and 66 years). N₂ fixation capacity on bryophytes was primarily driven by epiphytic cyanobacteria abundance rather than community composition. Most liverworts showed low colonization and N₂ fixation rates, and mosses did not exhibit consistent differences across life forms and habitat (saxicolous vs terricolous). We also found a clear relationship between cyanobacteria genera and the stages of ecological succession, but no relationship was found with host species identity. Glacier forelands in Tierra del Fuego show fast rates of soil transformation which imply large quantities of N inputs. Our results highlight the potential contribution of bryophyte-cyanobacteria associations to N accumulation during post-glacial primary succession and further describe the factors that drive N₂-fixation rates in post-glacial areas with very low N deposition.     

Light-Limited Growth Rate Modulates Nitrate Inhibition of Dinitrogen Fixation in the Marine Unicellular Cyanobacterium Crocosphaera watsonii

Biological N₂ fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO₃ ⁻). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N₂ fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N₂-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N₂ fixation. When growth (µ) was limited by low light (µ = 0.23 d⁻¹), short-term experiments indicated that 0.4 µM NH₄ ⁺ reduced N₂-fixation by ∼90% relative to controls without added NH₄ ⁺. In fast-growing, high-light-acclimated cultures (µ = 0.68 d⁻¹), 2.0 µM NH₄ ⁺ was needed to achieve the same effect. In long-term exposures to NO₃ ⁻, inhibition of N₂ fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO₃ ⁻ did not inhibit N₂-fixation rates in comparison with cultures growing on N₂ alone. Instead NO₃ ⁻ supported even faster growth, indicating that the cellular assimilation rate of N₂ alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO₃ ⁻ did not support faster growth rates but instead inhibited N₂-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N₂ fixation by fixed N sources based on the growth rate as controlled by light.     

The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen (¹⁵N) N₂ gas stocks with ¹⁵N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. ¹⁵N₂ gas is used to estimate N₂ fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled ¹⁵N₂ into organic matter. However, the microbial assimilation of bioavailable ¹⁵N-labeled N₂ gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N₂ fixation rates. ¹⁵N₂ gas procured from three major suppliers was analyzed for the presence of these ¹⁵N-contaminants. Substantial concentrations of ¹⁵N-contaminants were detected in four Sigma-Aldrich ¹⁵N₂ lecture bottles from two discrete batch syntheses. Per mole of ¹⁵N₂ gas, 34 to 1900 µmoles of ¹⁵N-ammonium, 1.8 to 420 µmoles of ¹⁵N-nitrate/nitrite, and ≥21 µmoles of ¹⁵N-nitrous oxide were detected. One ¹⁵N₂ lecture bottle from Campro Scientific contained ≥11 µmoles of ¹⁵N-nitrous oxide per mole of ¹⁵N₂ gas, and no detected ¹⁵N-nitrate/nitrite at the given experimental ¹⁵N₂ tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles ¹⁵N-nitrous oxide per mole ¹⁵N₂, and trace concentrations of ¹⁵N-ammonium and ¹⁵N-nitrate/nitrite. ¹⁵N₂ gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the ¹⁵N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N₂ fixation assays suggests that the degree of detected ¹⁵N-ammonium contamination could yield inferred N₂ fixation rates ranging from undetectable, <0.01 nmoles N L⁻¹ d⁻¹, to 530 nmoles N L⁻¹ d⁻¹, contingent on experimental conditions. These rates are comparable to, or greater than, N₂ fixation rates commonly detected in field assays. These results indicate that past reports of N₂ fixation should be interpreted with caution, and demonstrate that the purity of commercial ¹⁵N₂ gas must be ensured prior to use in future N₂ fixation rate determinations.     

Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Epiphytic Cyanobacteria on Chara vulgaris Are the Main Contributors to N2 Fixation in Rice Fields

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N₂ fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N₂ fixation rate associated with Chara was 27.53 kg of N ha⁻¹ crop⁻¹. The mean estimated N₂ fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha⁻¹ crop⁻¹; submerged parts of rice plants, 3.93 kg of N ha⁻¹ crop⁻¹; and roots, 0.28 kg of N ha⁻¹ crop⁻¹. Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% ± 4.4% and 6.2% ± 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 ± 28.0 and 4.0 ± 3.8 μg mg [dry weight] of Chara⁻¹ in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

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.Subject terms: Microbial ecology, Biogeochemistry, Stable isotope analysis     

Maize Yield Response to Water Supply and Fertilizer Input in a Semi-Arid Environment of Northeast China

Maize grain yield varies highly with water availability as well as with fertilization and relevant agricultural management practices. With a 311-A optimized saturation design, field experiments were conducted between 2006 and 2009 to examine the yield response of spring maize (Zhengdan 958, Zea mays L) to irrigation (I), nitrogen fertilization (total nitrogen, urea-46% nitrogen,) and phosphorus fertilization (P₂O₅, calcium superphosphate-13% P₂O₅) in a semi-arid area environment of Northeast China. According to our estimated yield function, the results showed that N is the dominant factor in determining maize grain yield followed by I, while P plays a relatively minor role. The strength of interaction effects among I, N and P on maize grain yield follows the sequence N+I >P+I>N+P. Individually, the interaction effects of N+I and N+P on maize grain yield are positive, whereas that of P+I is negative. To achieve maximum grain yield (10506.0 kg·ha⁻¹) for spring maize in the study area, the optimum application rates of I, N and P are 930.4 m³·ha⁻¹, 304.9 kg·ha⁻¹ and 133.2 kg·ha⁻¹ respectively that leads to a possible economic profit (EP) of 10548.4 CNY·ha⁻¹ (CNY, Chinese Yuan). Alternately, to obtain the best EP (10827.3 CNY·ha⁻¹), the optimum application rates of I, N and P are 682.4 m³·ha⁻¹, 241.0 kg·ha⁻¹ and 111.7 kg·ha⁻¹ respectively that produces a potential grain yield of 10289.5 kg·ha⁻¹.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Calorimetry of nitrogenase-mediated reductions in detached soybean nodules

Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N₂ fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.

There was major heat output by nodules for all of the nitrogenase substrates tested (H⁺, N₂, N₂O, and C₂H₂) further establishing the in vivo energy inefficiency of biological N₂ fixation. Exposure to a short burst of 100% O₂ partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H⁺ reductions was 171 ± 6 kilocalories per mole H₂ evolved in an Ar-O₂ atmosphere, that for N₂ fixation was 784 ± 26 kilocalories per mole H₂ evolved and N₂ fixed, and that for C₂H₂ reduction was 250 ± 12 kilocalories/mole C₂H₄ formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e⁻ is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N₂ fixation cost of 9.5 grams glucose per gram N₂ fixed or 3.8 grams C per gram N₂, which is in close agreement with earlier calculations based on nodular CO₂ production.

     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

HCO3− Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO₃⁻ fixation by these Thiothrix tufts was determined to be 14.0 ± 5.4 nmol of HCO₃⁻ per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO₃⁻ fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, α-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO₃⁻ fixation, 5 to 10 mM malate inhibited HCO₃⁻ fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO₃⁻ at rates as high as 29.9 ± 2.8 nmol of HCO₃⁻ per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO₃⁻ fixation.