Nitrogen fixation

The ability to exploit the atmospheric reservoir of nitrogen gas (or, at least, that fraction dissolved in water: at sea-level air-equilibrium, ~20 mg nitrogen L-1 at 0 °C, falling to ~11 mg L-1 at 20 °C) as a source of nutrient is exclusively associated with prokaryotes. This ability to 'fix' (reduce) elemental dinitrogen to ammonia is a widespread trait among obligate heterotrophic chemolithotrophic bacteria, the photosynthetic bacteria and the Cyanobacteria. Certain of the latter, most especially, some of the nostocalean genera, are the only members of phytoplankton to have this capacity. Nitrogen fixation may have been a crucial step in the evolution of autotrophy in an increasingly oxygenic atmosphere, because of the relative volatility and extreme sparseness of nitrogen in the lithosphere. Ammonia was also rare owing to its photolysis in an atmosphere relatively undefended against ultraviolet radiation. The early emergence of biological fixation, through the production of the dinitrogen reduc-tase enzyme, provided the first means of entry into ecosystems of large quantities of combined nitrogen (Falkowski, 2002). The enzyme catalyses the reduction of dinitrogen to ammonium using reductant produced via carbohydrate oxidation. Nitrogen fixation is a respiratory reaction:

Interestingly, dinitrogen reductases are based on iron-sulphur prosthetic groups that are redox sensitive: the enzymes operate only under strictly anaerobic conditions (as they did when they evolved). Nitrogen fixation is rapidly inactivated in the presence of oxygen (Yates, 1977). In order to fix nitrogen in an oxic ocean, the enzyme must be protected from poisoning by oxygen. As Paerl (1988) remarked, for compatibility between oxy-genic photosynthesis and anoxic nitrogen fixation to have developed represents a remarkable evolutionary achievement for the Cyanobacteria.

Until the 1960s, the nitrogen-fixing capability of Cyanobacteria had only been suspected from nitrogen budgets (e.g. Dugdale et al., 1959). The introduction of the acetylene-reduction assay for nitrogenase activity (Stewart et al., 1967) made it possible to investigate which species fixed nitrogen, under what conditions and at which locations. Among the freshwater Nostocales, fixation is confined to the heterocysts (sometimes called heterocytes). These are specialised cells differentiated at intervals along the vegetative filaments (Fay et al., 1968). Their thick walls defend the intracellular anaerobic conditions necessary for the enzyme function. They are differentiated in life from normal vegetative cells responding to nitrogen deficiency. Besides the thickening of the wall, the cells lose their blue-coloured phy-cocyanin. However, they retain a chlorophyll-based light-harvesting capacity, attached to a functional PS I and ferredoxin transfer pathway to NADP reduction (cf. Section 3.2.1) but the oxygen-evolving PS II is, of course, defunct (Wolk, 1982; Paerl, 1988).

Heterocysts are not permanent features. Natural populations of Anabaena, Aphanizomemon, etc. can increase to significant levels of biomass without producing heterocysts. Differentiation is a facultative response to falling external DIN concentrations, to the extent that their relative frequency (heterocysts : vegetative cells) has been taken by ecologists to be a sign of incipient nitrogen limitation (Horne and Commins, 1987; Reynolds, 1987a). Their induction and distribution are regulated genetically by DNA promoters (see Mann, 1995, for details). Most of the relevant observations on their production (reviewed in Reynolds, 1987a) report the incidence of increased heterocyst frequency, from <1 : 10 000 vegetative cells to as high as 1 : 10, in populations of Anabaena, Aphanizomenon and Nodularia, coincident with DIN concentrations falling below 300-350 mg m-3 (19-25 | M). The higher ratios are noted particularly at DIN concentrations <80 mg m-3 (<6 |M N). Given that these concentrations will, ostensibly, at least half-saturate the maximum rates of uptake of combined nitrogen and completely saturate nitrogen demand (see above), the sensitivity of heterocyst production to rather higher DIN concentrations is puzzling. One possible explanation is that the heterocyst production and, indeed, the nitrogenase activity that they accommodate are actually sensitive to the external concentrations of ammonium, which may represent much the smaller fraction of the total DIN pool and is also the one that is the more rapidly drawn down. This would also have to imply that the nitrogen-fixation response is a preferential reaction to low external levels of NH4.N (<0.5 |M N, or <7 mg N m-3) and not of nitrate. Direct sensitivity of nitrogenase production in Anabaena flos-aquae to ammonium concen trations has been demonstrated in the laboratory (Ohmori and Hattori, 1974; see also Kerby et al., 1987), albeit at higher levels. On the other hand, the isolates of non-nitrogen-fixing Cyanobacteria from nitrogen-deficient lakes (Merismopedia, Micro-cystis, Synechococcus) used in the experiments of Blomqvist et al. (1994), all responded much more positively to ammonium enrichment than they did to nitrate additions. They also responded less positively to nitrate additions than did plank-tic eukaryotes, including a Peridinium. Bearing in mind that the group evolved in an ammonium-scarce, nitrate-free, anoxic environment, a high affinity for ammonium nitrogen and a low-redox mechanism for its intracellular enhancement would appear to be useful adaptations. Both retain a relevance to survival and relative success of distinctive members of the group in modern environments that are extremely nitrogen-deficient or where relatively high phosphorus levels drive biomass accumulation of producers able to exploit extraneous sources of nitrogen.

Besides low external DIN concentrations and a low-redox, microaerophilous proximal environment, adequate nitrogen fixation remains dependent upon high electron transport energy as well as high rates of endogenous respiration (Paerl, 1988), driven (in this instance) by photosynthesis and good insolation. The low reactivity of N2 requires that large amounts of ATP and reducing power are invested in the nitrogenase reaction (Postgate, 1987). Nitrogen fixation also requires phosphorus: Stewart and Alexander (1971) showed that nitrogenase activity was steadily lost in cultures of heterocystous Anabaena and other nostocalean species transferred to P-free medium and was not restored without the addition of phosphate to the medium to a concentration equivalent to 5 mg P m-3 (~0.16 |M P). The availability of molybdenum and/or vanadium/iron for the core of the nitrogenase enzyme is biochemically essential to nitrogen fixation (Postgate, 1987; Rueter and Petersen, 1987). It cannot be assumed that low ambient nitrogen levels are automatically compensated by nitrogen fixation and the successful exploitation of such conditions by N2-fixing Cyanobacteria, without demonstrable satisfaction of the constraints imposed by light, phosphorus and micronutrient deficiencies.

Nitrogen fixation can occur, or has been induced, in other non-heterocystous genera of freshwater Cyanobacteria (Plectonema: Stewart and Lex, 1970; Gloeocapsa: Rippka et al., 1971). The maintenance of an oxygen-free microenvironment remains a paramount precondition. One way in which this can be achieved is through the dense adpositioning of trichomes into dense bundles or rafts (Carpenter and Price, 1976; Paerl, 1988). The effect is further enhanced by bathing filaments in mucilage containing reducing sul-phydryl groups (Sirenko et al., 1968). Nitrogen fixation also occurs among mat-forming littoral species of Oscillatoria but only during darkness when there is no photosynthetic oxygen generation (Bautista and Paerl, 1985). For many other common freshwater genera (Microcystis, Woroni-chinia, Gomphosphaeria), no such facility has been demonstrated.

In the marine phytoplankton, nitrogen fixers are represented by the non-heterocystous marine species of oscillatorialean genus of Tri-chodesmium. Each of the three recognised species has adopted the life habit of forming large macroscopic rafts, or flakes, of uniseriate filaments. These bundles were sufficiently prominent in the very clear tropical and subtropical seas where they mainly occur for early mariners to have named them 'sea sawdust' (given the reddish-brown accessory pigmentation of the flakes, the name is apposite, elegantly conveying a good description of their appearance). Living in environments of the Atlantic Ocean maintaining very low levels of inorganic combined nitrogen (often <1 |M DIN), Trichodesmium thiebau-tii nevertheless fixes nitrogen, aerobically and whilst photosynthesising, sufficient to satisfy the bulk of its nitrogen requirements (Carpenter and McCarthy, 1975). This metabolism is energetically expensive and relatively slow but is adequate to support Trichodesmium dominance over almost all other, nitrogen-starved phytoplankters (Carpenter and Romans, 1991; Zehr, 1995).

This being so, it has long been puzzling to ecologists why there are not more genera of nitrogen fixers in the nitrogen-deficient oceans or even why Trichodesmium is not more abun dant than it is and contributing a larger part to the oceanic turnover of nitrogen. Zehr's (1995) careful exploration of these questions confirmed the widespread occurrence among the Cyanobac-teria of the nitrogenase-encoding DNA but that its expression in nitrogenase activity was as limited as previously circumscribed. It is not known to be expressed among the picoplanktic Cyanobacteria but fixers were sometimes incorporated in the microaerophilic zones of sinking particulate clusters of marine snow. Expression of nitrogenase activity among other members of the Oscillatoriales is confined to microaerophilous conditions, which Trichodesmium is uniquely able to contrive through its own growth habit. It is not relatively abundant where more nutrients (including DIN) deny to Trichodesmium its dynamic advantage. As to why it is not more abundant in the low-DIN oceans, it was supposed, for a time, that micronutrient deficiencies (in particular, of iron and molybdenum) so interfere with nitrogen fixation that the otherwise obvious potential advantage that nitrogen fixation might confer is suppressed. In fact, as is now well known, relatively high-nitrogen, low-chlorophyll regions of the great oceans augur that the biomass capacity is constrained by micronutrient availability per se (see Section 4.5), which access to alternative exploitable sources of nitrogen fails to alleviate. Not even the diatom Hemiaulus, with its nitrogen-fixing endosymbiont, Richiella (Heinbokel, 1986), is able to gain much advantage over other species of the tropical gyres. The problem is still to satisfy the simultaneous requirements of nitrogen fixation. The ability to fix nitrogen really provides an advantage only in those parts of the sea where DIN is truly limiting and where energy, phosphorus and adequate sources of iron, molybdenum and vanadium are simultaneously sufficient to support it.

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