Temperature excludes N2-fixing heterocystous cyanobacteria in the tropical oceans

Whereas the non-heterocystous cyanobacteria Trichodesmium spp. are the dominant N2-fixing organisms in the tropical oceans, heterocystous species dominate N2 fixation in freshwater lakes and brackish environments such as the Baltic Sea. So far no satisfactory explanation for the absence of heterocystous cyanobacteria in the pelagic of the tropical oceans has been given, even though heterocysts would seem to represent an ideal strategy for protecting nitrogenase from being inactivated by O2, thereby enabling cyanobacteria to fix N2 and to perform photosynthesis simultaneously. Trichodesmium is capable of N2 fixation, apparently without needing to differentiate heterocysts. Here we show that differences in the temperature dependence of O2 flux, respiration and N2 fixation activity explain how Trichodesmium performs better than heterocystous species at higher temperatures. Our results also explain why Trichodesmium is not successful in temperate or cold seas. The absence of heterocystous cyanobacteria in the pelagic zone of temperate and cold seas, however, requires another explanation.     

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean's nitrogen inventory and primary productivity. Nitrogen-isotope data from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000?years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200?Tg?N?yr?1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and γ-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14?±?1?Tg?N?yr?1 and 24?±1?Tg?N?yr?1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103?±?8?Tg?N?yr?1 to 177?±?8?Tg?N?yr?1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.     

High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean

The availability of nitrogen is important in regulating biological productivity in marine environments. Deepwater nitrate has long been considered the major source of new nitrogen supporting primary production in oligotrophic regions of the open ocean, but recent studies have showed that biological N2 fixation has a critical role in supporting oceanic new production. Large colonial cyanobacteria in the genus Trichodesmium and the heterocystous endosymbiont Richelia have traditionally been considered the dominant marine N2 fixers, but unicellular diazotrophic cyanobacteria and bacterioplankton have recently been found in the picoplankton and nanoplankton community of the North Pacific central gyre, and a variety of molecular and isotopic evidence suggests that these unicells could make a major contribution to the oceanic N budget. Here we report rates of N2 fixation by these small, previously overlooked diazotrophs that, although spatially variable, can equal or exceed the rate of N2 fixation reported for larger, more obvious organisms. Direct measurements of 15N2 fixation by small diazotrophs in various parts of the Pacific Ocean, including the waters off Hawaii where the unicellular diazotrophs were first characterized, show that N2 fixation by unicellular diazotrophs can support a significant fraction of total new production in oligotrophic waters.     

A unifying framework for dinitrogen fixation in the terrestrial biosphere

Dinitrogen (N(2)) fixation is widely recognized as an important process in controlling ecosystem responses to global environmental change, both today and in the past; however, significant discrepancies exist between theory and observations of patterns of N(2) fixation across major sectors of the land biosphere. A question remains as to why symbiotic N(2)-fixing plants are more abundant in vast areas of the tropics than in many of the mature forests that seem to be nitrogen-limited in the temperate and boreal zones. Here we present a unifying framework for terrestrial N(2) fixation that can explain the geographic occurrence of N(2) fixers across diverse biomes and at the global scale. By examining trade-offs inherent in plant carbon, nitrogen and phosphorus capture, we find a clear advantage to symbiotic N(2) fixers in phosphorus-limited tropical savannas and lowland tropical forests. The ability of N(2) fixers to invest nitrogen into phosphorus acquisition seems vital to sustained N(2) fixation in phosphorus-limited tropical ecosystems. In contrast, modern-day temperatures seem to constrain N(2) fixation rates and N(2)-fixing species from mature forests in the high latitudes. We propose that an analysis that couples biogeochemical cycling and biophysical mechanisms is sufficient to explain the principal geographical patterns of symbiotic N(2) fixation on land, thus providing a basis for predicting the response of nutrient-limited ecosystems to climate change and increasing atmospheric CO(2).     

Phosphorus limitation of nitrogen fixation by Trichodesmium in the central Atlantic Ocean

Marine fixation of atmospheric nitrogen is believed to be an important source of biologically useful nitrogen to ocean surface waters, stimulating productivity of phytoplankton and so influencing the global carbon cycle. The majority of nitrogen fixation in tropical waters is carried out by the marine cyanobacterium Trichodesmium, which supplies more than half of the new nitrogen used for primary production. Although the factors controlling marine nitrogen fixation remain poorly understood, it has been thought that nitrogen fixation is limited by iron availability in the ocean. This was inferred from the high iron requirement estimated for growth of nitrogen fixing organisms and the higher apparent densities of Trichodesmium where aeolian iron inputs are plentiful. Here we report that nitrogen fixation rates in the central Atlantic appear to be independent of both dissolved iron levels in sea water and iron content in Trichodesmium colonies. Nitrogen fixation was, instead, highly correlated to the phosphorus content of Trichodesmium and was enhanced at higher irradiance. Furthermore, our calculations suggest that the structural iron requirement for the growth of nitrogen-fixing organisms is much lower than previously calculated. Although iron deficiency could still potentially limit growth of nitrogen-fixing organisms in regions of low iron availability-for example, in the subtropical North Pacific Ocean-our observations suggest that marine nitrogen fixation is not solely regulated by iron supply.     

Reduced nitrogen fixation in the glacial ocean inferred from changes in marine nitrogen and phosphorus inventories.

To explain the lower atmospheric CO2 concentrations during glacial periods, it has been suggested that the productivity of marine phytoplankton was stimulated by an increased flux of iron-bearing dust to the oceans. One component of this theory is that iron-an essential element/nutrient for nitrogen-fixing organisms-will increase the rate of marine nitrogen fixation, fuelling the growth of other marine phytoplankton and increasing CO2 uptake. Here we present data that questions this hypothesis. From a sediment core off the northwestern continental margin of Mexico, we show that denitrification and phosphorite formation-processes that occur in oxygen-deficient upwelling regions, removing respectively nitrogen and phosphorus from the ocean-declined in glacial periods, thus increasing marine inventories of nitrogen and phosphorus. But increases in phosphorus were smaller and less rapid, leading to increased N/P ratios in the oceans. Acknowledging that phytoplankton require nitrogen and phosphorus in constant proportions, the Redfield ratio, and that N/P ratios greater than the Redfield ratio are likely to suppress nitrogen fixation, we suggest therefore that marine productivity did not increase in glacial periods in response to either increased nutrient inventories or greater iron supply.     

Spatial coupling of nitrogen inputs and losses in the ocean

Nitrogen fixation is crucial for maintaining biological productivity in the oceans, because it replaces the biologically available nitrogen that is lost through denitrification. But, owing to its temporal and spatial variability, the global distribution of marine nitrogen fixation is difficult to determine from direct shipboard measurements. This uncertainty limits our understanding of the factors that influence nitrogen fixation, which may include iron, nitrogen-to-phosphorus ratios, and physical conditions such as temperature. Here we determine nitrogen fixation rates in the world's oceans through their impact on nitrate and phosphate concentrations in surface waters, using an ocean circulation model. Our results indicate that nitrogen fixation rates are highest in the Pacific Ocean, where water column denitrification rates are high but the rate of atmospheric iron deposition is low. We conclude that oceanic nitrogen fixation is closely tied to the generation of nitrogen-deficient waters in denitrification zones, supporting the view that nitrogen fixation stabilizes the oceanic inventory of fixed nitrogen over time.     

Dinitrogen fixation by a thermophilic methanogenic bacterium

Methanogenic bacteria are known to use NH+4 as a nitrogen source for growth. Previous work with an impure methanogenic culture suggested that a methanogen might fix atmospheric dinitrogen as a nitrogen source, but no further work on this phenomenon has been documented. We have now examined the use of N2 by Methanococcus thermolithotrophicus and find that the organism can grow well, with multiple transfers, in medium having N2 as the source of nitrogen. Control cultures without N2 and containing less than 0.1 mM NH+4 do not grow. Growth yields with N2 are on the average one-third those with NH+4, suggesting that, as in other nitrogen-fixing organisms, this bacterium requires a large amount of ATP for the reduction to occur. After growing in NH+4-containing medium, a long lag is observed before growth begins with N2 as the nitrogen source; the NH+4 levels must be very low for growth to begin. Cells grown in N2-fixing conditions reduce acetylene to ethylene. The discovery of a nitrogen-fixing archaebacterium has important implications for studies on the evolution of nitrogenase, and the fact that M. thermolithotrophicus nitrogenase is active at 64 degrees C suggests that a novel enzyme is involved.     

N2 production by the anammox reaction in the anoxic water column of Golfo Dulce,Costa Rica

In oxygen-depleted zones of the open ocean, and in anoxic basins and fjords, denitrification (the bacterial reduction of nitrate to give N2) is recognized as the only significant process converting fixed nitrogen to gaseous N2. Primary production in the oceans is often limited by the availability of fixed nitrogen such as ammonium or nitrate, and nitrogen-removal processes consequently affect both ecosystem function and global biogeochemical cycles. It was recently discovered that the anaerobic oxidation of ammonium with nitrite--the 'anammox' reaction, performed by bacteria--was responsible for a significant fraction of N2 production in some marine sediments. Here we show that this reaction is also important in the anoxic waters of Golfo Dulce, a 200-m-deep coastal bay in Costa Rica, where it accounts for 19-35% of the total N2 formation in the water column. The water-column chemistry in Golfo Dulce is very similar to that in oxygen-depleted zones of the oceans--in which one-half to one-third of the global nitrogen removal is believed to occur. We therefore expect the anammox reaction to be a globally significant sink for oceanic nitrogen.     

Buckminsterfullerenes: a non-metal system for nitrogen fixation

In all nitrogen-fixation processes known so far--including the industrial Haber-Bosch process, biological fixation by nitrogenase enzymes and previously described homogeneous synthetic systems--the direct transformation of the stable, inert dinitrogen molecule (N2) into ammonia (NH3) relies on the powerful redox properties of metals. Here we show that nitrogen fixation can also be achieved by using a non-metallic buckminsterfullerene (C60) molecule, in the form of a water-soluble C60:gamma-cyclodextrin (1:2) complex, and light under nitrogen at atmospheric pressure. This metal-free system efficiently fixes nitrogen under mild conditions by making use of the redox properties of the fullerene derivative.     

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the world's largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific 'ladderane' membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.     

Nitrogen limitation constrains sustainability of ecosystem response to CO2

Enhanced plant biomass accumulation in response to elevated atmospheric CO2 concentration could dampen the future rate of increase in CO2 levels and associated climate warming. However, it is unknown whether CO2-induced stimulation of plant growth and biomass accumulation will be sustained or whether limited nitrogen (N) availability constrains greater plant growth in a CO2-enriched world. Here we show, after a six-year field study of perennial grassland species grown under ambient and elevated levels of CO2 and N, that low availability of N progressively suppresses the positive response of plant biomass to elevated CO2. Initially, the stimulation of total plant biomass by elevated CO2 was no greater at enriched than at ambient N supply. After four to six years, however, elevated CO2 stimulated plant biomass much less under ambient than enriched N supply. This response was consistent with the temporally divergent effects of elevated CO2 on soil and plant N dynamics at differing levels of N supply. Our results indicate that variability in availability of soil N and deposition of atmospheric N are both likely to influence the response of plant biomass accumulation to elevated atmospheric CO2. Given that limitations to productivity resulting from the insufficient availability of N are widespread in both unmanaged and managed vegetation, soil N supply is probably an important constraint on global terrestrial responses to elevated CO2.     

Phosphonate utilization by the globally important marine diazotroph Trichodesmium

The factors that control the growth and nitrogen fixation rates of marine diazotrophs such as Trichodesmium have been intensively studied because of the role that these processes have in the global cycling of carbon and nitrogen, and in the sequestration of carbon to the deep sea. Because the phosphate concentrations of many ocean gyres are low, the bioavailability of the larger, chemically heterogeneous pool of dissolved organic phosphorus could markedly influence Trichodesmium physiology. Here we describe the induction, by phosphorus stress, of genes from the Trichodesmium erythraeum IMS101 genome that are predicted to encode proteins associated with the high-affinity transport and hydrolysis of phosphonate compounds by a carbon-phosphorus lyase pathway. We show the importance of these genes through expression analyses with T. erythraeum from the Sargasso Sea. Phosphonates are known to be present in oligotrophic marine systems, but have not previously been considered to be bioavailable to marine diazotrophs. The apparent absence of genes encoding a carbon-phosphorus lyase pathway in the other marine cyanobacterial genomes suggests that, relative to other phytoplankton, Trichodesmium is uniquely adapted for scavenging phosphorus from organic sources. This adaptation may help to explain the prevalence of Trichodesmium in low phosphate, oligotrophic systems.     

大豆和花生根瘤菌氢酶的研究

根瘤菌在共生固氮过程中因放H2所消耗的能量约占固所氮总能量的40%-6%。吸氢酶则能回收和利用固氮过程所放的H2,养活能量损失,从而提高共生固氮效率。在厌氧条件下,加入防止酶蛋白聚合的试剂,利用DEAE-纤维素和Sephacry S-200柱层析,从自养性大豆根瘤菌和花生根瘤菌类菌体中分离并提纯膜结合态氢酶。纯化的两种氢酶表现相近的分子特征：均含有大（60kD,65kD)、小（30kD,35kD)两个亚基,均为NiFe-氢酶,并且有较高的吸H2活性。大豆根瘤菌氢酶的纯酶组分不含Cyt b599。花生根瘤菌L8-3菌株能进行化能自养生长,诱导出高吸H2活性。根瘤菌的吸H2能明显提高固氮活性。从具有高吸H2活性的花生根瘤菌中分离并克隆吸氢基因,采用PCR和探针杂交技术,获得含有吸氢基因的质粒pZ-55。利用多种限制性内切酶构建了质粒pZ-55的物理图谱。通过三亲本杂交,将含吸氢基因的重组质粒转移到不吸H2的花生和毛豆根瘤菌中,所获得的结合株在自生和共生条件下均表达吸H2活性。以结合株接种大田花生,获得的共生根瘤的吸H2活性比接种受体株提高4倍,花生叶片和种子的含N量、产量分别提高1.7%、8.9%和9.6%。     

Submerged culture of Magne-tospirillum gryphiswaldense under N2-fixing condition and regulation of activity of nitrogen fixation

A submerged culture technique for Magnetospirillum gryphiswaldense under the nitrogen-fixing condition (microaerobic and N-limited) was set up. In N-limited medium with Na-lactate as a sole carbon source, the optical density (A600 nm) and activity of nitrogen fixation of cells were 1.3 and 217 nmol of ethylene produced per hour per A600nm respectively within 21 h by three times of feeds. The pH and temperature were controlled at 7.2 and 30℃ respectively, and the oxygen concentration was controlled by sparging with N2 containing 0.4%-0.8% of O2. The activity of nitrogen fixation of cells was obviously inhibited by oxygen and ammonium. It indicated that the posttranslational regulation of nitrogenase existed in M. gryphiswaldense.     

Energy requirement for symbiotic nitrogen fixation

Comparing the energy required by legumes for symbiotic nitrogen fixation with that of assimilation of nitrate, Gibson concluded the costs to be about the same. About 15% of net photosynthetic production by the plant may be used in meeting its nitrogen requirements. If energy for the fixation of nitrogen symbiotically and that for the assimilation of NH4+ or NO3- from the soil solution are both provided by the chemical products of photosynthesis, then the CO2 respired in supplying that energy must contribute to the total efflux of CO2 from the plant in the dark. McCree and Thornley have discussed methods of partitioning the dark CO2 efflux into a growth (synthesis) and a maintenance component. Both nitrogen fixation and nitrogen assimilation can be expected to contribute to the CO2 flux associated with synthesis. If Gibson is correct, nodulated plants using only symbiotically fixed nitrogen should have the same growth coefficient as non-nodulated plants supplied with exogenous mineral nitrogen when grown in the same conditions. I have examined this hypothesis using a modification of the method used by McCree with Trifolium subterraneum L. cultivar Woogenellup as test material.     

Slowdown of the meridional overturning circulation in the upper Pacific Ocean

Decadal temperature fluctuations in the Pacific Ocean have a significant effect on marine ecosystems and the climate of North America. The physical mechanisms responsible for these fluctuations are poorly understood. Some theories ascribe a central role to the wind-driven meridional overturning circulation between the tropical and subtropical oceans. Here we show, from observations over the past 50 years, that this overturning circulation has been slowing down since the 1970s, causing a decrease in upwelling of about 25% in an equatorial strip between 9 degrees N and 9 degrees S. This reduction in equatorial upwelling of relatively cool water, from 47 x 10(6) to 35 x 10(6) m3 s(-1), is associated with a rise in equatorial sea surface temperatures of about 0.8 degrees C. Another effect of the slowing circulation is a reduction in the outgassing of CO2 from the equatorial Pacific Ocean-at present the largest oceanic source of carbon dioxide to the atmosphere.     

Latitudinal variations in plankton delta 13C: implications for CO2 and productivity in past oceans

The stable-carbon isotopic composition of marine organic material has varied significantly over geological time, and reflects significant excursions in the isotopic fractionation associated with the uptake of carbon by marine biota. For example, low 13C/12C in Cretaceous sediments has been attributed to elevated atmospheric (and hence oceanic) CO2 partial pressures. A similar depletion in 13C present-day Antarctic plankton has also been ascribed to high CO2 availability. We report, however, that this high-latitude isotope depletion develops at CO2 partial pressures (pCO2 levels) that are often below that of the present atmosphere (340 microatmospheres) , and usually below that of equatorial upwelling systems (> 340 microatmospheres). Nevertheless, because of the much lower water temperatures and, hence, greater CO2 solubility at high latitude, the preceding pCO2 measurements translate into Antarctic surface-water CO2 (aq) concentrations that are as much as 2.5 times higher than in equatorial waters. We calculate that an oceanic pCO2 level of > 800 microatmospheres (over twice the present atmospheric pCO2) in a warmer low-latitude Cretaceous ocean would have been required to produce the plankton 13C depletion preserved in Cretaceous sediments.     

Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.     

A possible nitrogen crisis for Archaean life due to reduced nitrogen fixation by lightning.

Nitrogen is an essential element for life and is often the limiting nutrient for terrestrial ecosystems. As most nitrogen is locked in the kinetically stable form, N2, in the Earth's atmosphere, processes that can fix N2 into biologically available forms-such as nitrate and ammonia-control the supply of nitrogen for organisms. On the early Earth, nitrogen is thought to have been fixed abiotically, as nitric oxide formed during lightning discharge. The advent of biological nitrogen fixation suggests that at some point the demand for fixed nitrogen exceeded the supply from abiotic sources, but the timing and causes of the onset of biological nitrogen fixation remain unclear. Here we report an experimental simulation of nitrogen fixation by lightning over a range of Hadean (4.5-3.8 Gyr ago) and Archaean (3.8-2.5 Gyr ago) atmospheric compositions, from predominantly carbon dioxide to predominantly dinitrogen (but always without oxygen). We infer that, as atmospheric CO2 decreased over the Archaean period, the production of nitric oxide from lightning discharge decreased by two orders of magnitude until about 2.2 Gyr. After this time, the rise in oxygen (or methane) concentrations probably initiated other abiotic sources of nitrogen. Although the temporary reduction in nitric oxide production may have lasted for only 100 Myr or less, this was potentially long enough to cause an ecological crisis that triggered the development of biological nitrogen fixation.