Benthic N2 fixation in coral reefs and the potential effects of human‐induced environmental change

Tropical coral reefs are among the most productive and diverse ecosystems, despite being surrounded by ocean waters where nutrients are in short supply. Benthic dinitrogen (N₂) fixation is a significant internal source of “new” nitrogen (N) in reef ecosystems, but related information appears to be sparse. Here, we review the current state (and gaps) of knowledge on N₂ fixation associated with coral reef organisms and their ecosystems. By summarizing the existing literature, we show that benthic N₂ fixation is an omnipresent process in tropical reef environments. Highest N₂ fixation rates are detected in reef‐associated cyanobacterial mats and sea grass meadows, clearly showing the significance of these functional groups, if present, to the input of new N in reef ecosystems. Nonetheless, key benthic organisms such as hard corals also importantly contribute to benthic N₂ fixation in the reef. Given the usually high coral coverage of healthy reef systems, these results indicate that benthic symbiotic associations may be more important than previously thought. In fact, mutualisms between carbon (C) and N₂ fixers have likely evolved that may enable reef communities to mitigate N limitation. We then explore the potential effects of the increasing human interferences on the process of benthic reef N₂ fixation via changes in diazotrophic populations, enzymatic activities, or availability of benthic substrates favorable to these microorganisms. Current knowledge indicates positive effects of ocean acidification, warming, and deoxygenation and negative effects of increased ultraviolet radiation on the amount of N fixed in coral reefs. Eutrophication may either boost or suppress N₂ fixation, depending on the nutrient becoming limiting. As N₂ fixation appears to play a fundamental role in nutrient‐limited reef ecosystems, these assumptions need to be expanded and confirmed by future research efforts addressing the knowledge gaps identified in this review.     

Revisiting RuBisCO

Since the discovery of its role in the CO₂ fixation reaction in photosynthesis, RuBisCO has been one of the most extensively researched enzymes in the fields of biochemistry, molecular biology, and molecular genetics as well as conventional plant physiology, agricultural chemistry, and crop science. In addition, the RuBisCO and RuBisCO-like genes of more than 2000 organisms have been sequenced during the past 20 years. During the course of those studies, the origin of the RuBisCO gene began to be discussed. Recent studies have reported that the RuBisCO gene emerged in methanogenic bacteria long before photosynthetic organisms appeared. The origin of similar early genes might have allowed this gene to overcome changes in global environments during ancient and recent eras and to participate in the fixation of 200 GT of CO₂ annually. In this review, I focus on several points that have not been discussed at length in the literature thus far.     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Incorporation of Different N Sources and Light Response Curves of Nitrogenase and Photosynthesis by Cyanobacterial Blooms from Rice Fields

In this work, we estimate the contributions of the different sources of N incorporated by two N₂-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N₂-fixing cyanobacterial blooms in rice fields comes from N₂ fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N₂ fixation (only about 33–41% of the N incorporated came from N₂ fixation). Our results on the photodependence of C and N₂ fixation indicate that in both cyanobacterial blooms, N₂ fixation showed a steeper initial slope (α) and was saturated with less irradiance than C fixation, suggesting that N₂ fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N₂ fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.     

Diversity of <Emphasis Type="Italic">nifH</Emphasis> gene amplified from plankton-community DNA in a shallow eutrophic lake (Lake Donghu,Wuhan, China)

Biological nitrogen fixation (BNF) is one of the major nitrogen inputs into the biosphere, and the nitrogenase iron protein (nifH) gene plays important roles in regulating the molecular nitrogen (N₂) fixation process. The nifH gene has also been extensively used to study the diversity and function of nitrogen-fixing microorganisms. In this study, we investigated the diversity of the nifH gene by culture-independent methods to analysis the planktonic nitrogen-fixing organisms in Lake Donghu, Wuhan, the largest urban lake in China. Results indicate that nifH gene sequences cloned from planktonic-community DNA showed high similarity to the uncultured cyanobacterial sequences deposited in the GenBank database. Phylogenetic analysis on the basis of the translated amino acid sequences further showed that most nifH clones were closely related to the reported cyanobacterial nifH gene sequences. Results also indicate that there are similar planktonic nitrogen-fixing organisms in the relatively independent areas of Lake Donghu, even though different regions showed a wide gradient in trophic status. These and other observations led us to believe that studies on nifH gene diversity and expression will increase our ability to understand the ecological function of target nitrogen-fixing groups in aquatic ecosystems.     

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecosystem nitrogen fixation throughout the snow‐free period in subarctic tundra: effects of willow and birch litter addition and warming

Nitrogen (N) fixation in moss‐associated cyanobacteria is one of the main sources of available N for N‐limited ecosystems such as subarctic tundra. Yet, N₂ fixation in mosses is strongly influenced by soil moisture and temperature. Thus, temporal scaling up of low‐frequency in situ measurements to several weeks, months or even the entire growing season without taking into account changes in abiotic conditions cannot capture the variation in moss‐associated N₂ fixation. We therefore aimed to estimate moss‐associated N₂ fixation throughout the snow‐free period in subarctic tundra in field experiments simulating climate change: willow (Salix myrsinifolia) and birch (Betula pubescens spp. tortuosa) litter addition, and warming. To achieve this, we established relationships between measured in situ N₂ fixation rates and soil moisture and soil temperature and used high‐resolution measurements of soil moisture and soil temperature (hourly from May to October) to model N₂ fixation. The modelled N₂ fixation rates were highest in the warmed (2.8 ± 0.3 kg N ha^?1) and birch litter addition plots (2.8 ± 0.2 kg N ha^?1), and lowest in the plots receiving willow litter (1.6 ± 0.2 kg N ha^?1). The control plots had intermediate rates (2.2 ± 0.2 kg N ha^?1). Further, N₂ fixation was highest during the summer in the warmed plots, but was lowest in the litter addition plots during the same period. The temperature and moisture dependence of N₂ fixation was different between the climate change treatments, indicating a shift in the N₂ fixer community. Our findings, using a combined empirical and modelling approach, suggest that a longer snow‐free period and increased temperatures in a future climate will likely lead to higher N₂ fixation rates in mosses. Yet, the consequences of increased litter fall on moss‐associated N₂ fixation due to shrub expansion in the Arctic will depend on the shrub species’ litter traits.     

DIAZOTROPHIC GROWTH OF THE MARINE CYANOBACTERIUM TRICHODESMIUM IMS101 IN CONTINUOUS CULTURE: EFFECTS OF GROWTH RATE ON N2‐FIXATION RATE,BIOMASS, AND C:N:P STOICHIOMETRY1

Trichodesmium N₂ fixation has been studied for decades in situ and, recently, in controlled laboratory conditions; yet N₂‐fixation rate estimates still vary widely. This variance has made it difficult to accurately estimate the input of new nitrogen (N) by Trichodesmium to the oligotrophic gyres of the world ocean. Field and culture studies demonstrate that trace metal limitation, phosphate availability, the preferential uptake of combined N, light intensity, and temperature may all affect N₂ fixation, but the interactions between growth rate and N₂ fixation have not been well characterized in this marine diazotroph. To determine the effects of growth rate on N₂ fixation, we established phosphorus (P)–limited continuous cultures of Trichodesmium, which we maintained at nine steady‐state growth rates ranging from 0.27 to 0.67 d^?1. As growth rate increased, biomass (measured as particulate N) decreased, and N₂‐fixation rate increased linearly. The carbon to nitrogen ratio (C:N) varied from 5.5 to 6.2, with a mean of 5.8 ± 0.2 (mean ± SD, N = 9), and decreased significantly with growth rate. The N:P ratio varied from 23.4 to 45.9, with a mean of 30.5 ± 6.6 (mean ± SD, N = 9), and remained relatively constant over the range of growth rates studied. Relative constancy of C:N:P ratios suggests a tight coupling between the uptake of these three macronutrients and steady‐state growth across the range of growth rates. Our work demonstrates that growth rate must be considered when planning studies of the effects of environmental factors on N₂ fixation and when modeling the impact of Trichodesmium as a source of new N to oligotrophic regions of the ocean.     

Dinitrogen fixation in the world's oceans

The surface water of themarine environment has traditionally beenviewed as a nitrogen (N) limited habitat, andthis has guided the development of conceptualbiogeochemical models focusing largely on thereservoir of nitrate as the critical source ofN to sustain primary productivity. However,selected groups of Bacteria, includingcyanobacteria, and Archaea canutilize dinitrogen (N₂) as an alternativeN source. In the marine environment, thesemicroorganisms can have profound effects on netcommunity production processes and can impactthe coupling of C-N-P cycles as well as the netoceanic sequestration of atmospheric carbondioxide. As one component of an integrated Nitrogen Transport and Transformations project, we have begun to re-assess ourunderstanding of (1) the biotic sources andrates of N₂ fixation in the world'soceans, (2) the major controls on rates ofoceanic N₂ fixation, (3) the significanceof this N₂ fixation for the global carboncycle and (4) the role of human activities inthe alteration of oceanic N₂ fixation. Preliminary results indicate that rates ofN₂ fixation, especially in subtropical andtropical open ocean habitats, have a major rolein the global marine N budget. Iron (Fe)bioavailability appears to be an importantcontrol and is, therefore, critical inextrapolation to global rates of N₂fixation. Anthropogenic perturbations mayalter N₂ fixation in coastal environmentsthrough habitat destruction and eutrophication,and open ocean N₂ fixation may be enhancedby warming and increased stratification of theupper water column. Global anthropogenic andclimatic changes may also affect N₂fixation rates, for example by altering dustinputs (i.e. Fe) or by expansion ofsubtropical boundaries. Some recent estimatesof global ocean N₂ fixation are in therange of 100–200 Tg N (1–2 × 10¹⁴ g N)yr^–1, but have large uncertainties. Theseestimates are nearly an order of magnitudegreater than historical, pre-1980 estimates,but approach modern estimates of oceanicdenitrification.     

Significant nonsymbiotic nitrogen fixation in Patagonian ombrotrophic bogs

Nitrogen (N) nutrition in pristine peatlands relies on the natural input of inorganic N through atmospheric deposition or biological dinitrogen (N₂) fixation. However, N₂ fixation and its significance for N cycling, plant productivity, and peat buildup are mostly associated with the presence of Sphagnum mosses. Here, we report high nonsymbiotic N₂‐fixation rates in two pristine Patagonian bogs with diversified vegetation and natural N deposition. Nonsymbiotic N₂ fixation was measured in samples from 0 to 10, 10 to 20, and 40 to 50 cm depth using the ¹⁵N₂ assay as well as the acetylene reduction assay (ARA). The ARA considerably underestimated N₂ fixation and can thus not be recommended for peatland studies. Based on the ¹⁵N₂ assay, high nonsymbiotic N₂‐fixation rates of 0.3–1.4 μmol N₂ g^?1 day^?1 were found down to 50 cm under micro‐oxic conditions (2 vol.%) in samples from plots covered by Sphagnum magellanicum or by vascular cushion plants, latter characterized by dense and deep aerenchyma roots. Peat N concentrations point to greater potential of nonsymbiotic N₂ fixation under cushion plants, likely because of the availability of easily decomposable organic compounds and oxic conditions in the rhizosphere. In the Sphagnum plots, high N₂ fixation below 10 cm depth rather reflects the potential during dry periods or low water level when oxygen penetrates the top peat layer and triggers peat mineralization. Natural abundance of the ¹⁵N isotope of live Sphagnum (5.6 δ‰) from 0 to 10 cm points to solely N uptake from atmospheric deposition and nonsymbiotic N₂ fixation. A mean ¹⁵N signature of ?0.7 δ‰ of peat from the cushion plant plots indicates additional N supply from N mineralization. Our findings suggest that nonsymbiotic N₂ fixation overcomes N deficiency in different vegetation communities and has great significance for N cycling and peat accumulation in pristine peatlands.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

A comparative study of nitrogen fixation by the Anabaena-Azolla symbiosis and free-living populations of Anabaena spp. in Lake Ngahawa,New Zealand

Summary The symbiotic fern Azolla filiculoides var. rubra, which contains a blue-green nitrogen fixing alga Anabaena azollae, fixed 164 Kg N·ha^-1·ann^-1 in the littoral zone of a small eutrophic lake. Associated planktonic Anabaena spp. blooms, dominated by Anabaena spiroides, fixed 29.5Kg N·ha^-1·ann^-1. Nitrogen fixation in both organisms was not obviously related to ambient dissolved inorganic nitrogen levels. By comparing ¹⁵N–N₂ and acetylene reduction techniques, we determined a ratio of 3 moles C₂H₂ reduced to 1 mole of N₂ fixed. Combining this with results from one diurnal investigation, it was estimated that 24% of the total daily fixation by Azolla occurred at night. Highest nitrogen fixation rates in Azolla occurred when plant density was lowest. Nitrogen fixation by planktonic Anabaena spp. generally paralleled changes in biomass. Frond breakage due to wind caused a decrease in Azolla nitrogen fixation and growth which was followed by a bloom of planktonic Anabaena spp. A second Anabaena spp. bloom was instrumental in the summer decline of Azolla. Maximum growth and nitrogen fixation of both organisms did not occur simultaneously. If physical disruption to the Azolla mat does not occur, it is likely that growth of the population would continue throughout the year.This work was completed at the Department of Scientific and Industrial Research, Freshwater Section, PO Box 415, Taupo, New Zealand, with partial assistance of N.S.F. Grant BMS-74-20745 to C.R. Goldman     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

Bioavailability of mineral-associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

Life on Earth depends on N₂-fixing microbes to make ammonia from atmospheric N₂ gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N₂ fixation was once believed to have evolved during the Archean-Proterozoic times using Fe as a cofactor. However, δ¹⁵N values of paleo-ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo-oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral-associated trace metals to a model N₂-fixing bacterium Azotobacter vinelandii. N₂ fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N₂ fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N₂ fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral-associated trace metals are bioavailable as cofactors of nitrogenases to support N₂ fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox.     

How do vascular plants perform photosynthesis in extreme environments? An integrative ecophysiological and biochemical story

In this work, we review the physiological and molecular mechanisms that allow vascular plants to perform photosynthesis in extreme environments, such as deserts, polar and alpine ecosystems. Specifically, we discuss the morpho/anatomical, photochemical and metabolic adaptive processes that enable a positive carbon balance in photosynthetic tissues under extreme temperatures and/or severe water‐limiting conditions in C₃ species. Nevertheless, only a few studies have described the in situ functioning of photoprotection in plants from extreme environments, given the intrinsic difficulties of fieldwork in remote places. However, they cover a substantial geographical and functional range, which allowed us to describe some general trends. In general, photoprotection relies on the same mechanisms as those operating in the remaining plant species, ranging from enhanced morphological photoprotection to increased scavenging of oxidative products such as reactive oxygen species. Much less information is available about the main physiological and biochemical drivers of photosynthesis: stomatal conductance (g_s), mesophyll conductance (g_m) and carbon fixation, mostly driven by RuBisCO carboxylation. Extreme environments shape adaptations in structures, such as cell wall and membrane composition, the concentration and activation state of Calvin–Benson cycle enzymes, and RuBisCO evolution, optimizing kinetic traits to ensure functionality. Altogether, these species display a combination of rearrangements, from the whole‐plant level to the molecular scale, to sustain a positive carbon balance in some of the most hostile environments on Earth.     

Nitrogen sources for neotropical nasute termites: Fixation and selective foraging

Summary Studies with Nasutitermes and Rhynchotermes (Isoptera, Nasutitermitinae) in a Costa Rican rainforest suggest that nitrogen fixation by gut symbionts may play a significant role in termite nutrition. Leaf-litter feeders ingest more and fix less nitrogen than do wood-litter feeders; both species feed preferentially on N-rich foods in their foraging repertoire. Unusually high N₂ fixation (acetylene reduction) rates were found in Nasutitermes soldiers, thereby reducing their nutritional dependence on workers. Finally, N₂ fixation rates of termites diminished rapidly within 24 h after removal of a colony from the field, underlining the importance of conducting future N₂ fixation studies under field conditions.