The cyanobacterial nitrogen fixation paradox in natural waters.

Nitrogen fixation, the enzymatic conversion of atmospheric N (N 2) to ammonia (NH 3), is a microbially mediated process by which "new" N is supplied to N-deficient water bodies. Certain bloom-forming cyanobacterial species are capable of conducting N 2 fixation; hence, they are able to circumvent N limitation in these waters. However, this anaerobic process is highly sensitive to oxygen, and since cyanobacteria produce oxygen in photosynthesis, they are faced with a paradoxical situation, where one critically important (for supporting growth) biochemical process is inhibited by another. N 2-fixing cyanobacterial taxa have developed an array of biochemical, morphological, and ecological adaptations to minimize the "oxygen problem"; however, none of these allows N 2 fixation to function at a high enough efficiency so that it can supply N needs at the ecosystem scale, where N losses via denitrification, burial, and advection often exceed the inputs of "new" N by N 2 fixation. As a result, most marine and freshwater ecosystems exhibit chronic N limitation of primary production. Under conditions of perpetual N limitation, external inputs of N from human sources (agricultural, urban, and industrial) play a central role in determining ecosystem fertility and, in the case of N overenrichment, excessive primary production or eutrophication. This points to the importance of controlling external N inputs (in addition to traditional phosphorus controls) as a means of ensuring acceptable water quality and safe water supplies. Nitrogen fixation, the enzymatic conversion of atmospheric N 2 to ammonia (NH 3) is a  microbially-mediated process by which "new" nitrogen is supplied to N-deficient water bodies.  Certain bloom-forming cyanobacterial species are capable of conducting N 2 fixation; hence they are able to circumvent nitrogen limitation in these waters. However, this anaerobic process is highly sensitive to oxygen, and since cyanobacteria produce oxygen in photosynthesis, they are faced with a paradoxical situation, where one critically-important (for supporting growth) biochemical process is inhibited by another. Diazotrophic cyanobacterial taxa have developed an array of biochemical, morphological and ecological adaptations to minimize the "oxygen problem"; however, none of these allows N 2 fixation to function at a high enough efficiency so that it can supply N needs at the ecosystem scale, where N losses via denitrification, burial and advection often exceed the inputs of "new" N by N 2 fixation.  As a result, most marine and freshwater ecosystems exhibit chronic N-limitation of primary production.  Under conditions of perpetual N limitation, external inputs of N from human sources (agricultural, urban, industrial) play a central role in determining ecosystem fertility and in the case of N-overenrichment, excessive primary production, or eutrophication. This points to the importance of controlling external N inputs (in addition to traditional phosphorus controls) as a means of ensuring acceptable water quality and safe water supplies.

n class="Chemical">Nitrogen fixationpan>, the biochemical conpan>versionpan> of “inert” atmospheric N (N 2) to biologically available pan> class="Chemical">ammonia (NH 3), is a microbially mediated process of global significance because it provides “new” N to aquatic ecosystems in which biological production is often controlled by N availability [1, 2]. N 2 fixation is an anaerobic process carried out by specific prokaryotes, including heterotrophic and chemolithotrophic bacteria and some cyanobacteria (blue-green algae) [3]. The process likely evolved during the oxygen (O 2)-devoid Precambrian period some 2+ billion years ago [4, 5]. Of the N 2-fixing microbial taxa, the cyanobacteria are of particular biogeochemical and ecological interest because they were also the first O 2-evolving photosynthetic organisms on Earth [6]; their proliferation during this period is thought to be an evolutionary “milestone” because it led to the generation of an O 2-rich atmosphere, a prerequisite for the evolution of O 2-requiring fungi, bacteria, animals, and higher plant species on our planet [6].

n class="Chemical">Ironically, the developmenpan>t of anpan> O 2-rich atmosphere, hydrosphere, anpan>d pedosphere conpan>stituted a formidable biochemical challenpan>ge for the cyanpan>obacteria because, while they were capable of fixing N 2, the process had to be conpan>fined to anpan> O 2-free micro-enpan>vironment [7]. This requirement posed a serious dilemma, especially for aquatic cyanobacteria, because they require illuminated conditions in surface waters, but the high ambient O 2 levels produced by photosynthesis in these waters also represents an environmental barrier to O 2-sensitive N 2 fixation. Over their long evolutionary history, cyanobacteria have developed biochemical and structural adaptations as well as biotic associations in order to optimize N 2 fixation while relying on oxygenic photosynthesis to provide energy and organic carbon (C) compounds to support metabolism and growth. The adaptions include (1) confining N 2 fixation to night-time when photosynthesis is “turned off”, (2) forming colonies and aggregates to reduce illumination and form low-O 2 “microzones”, (3) participating as endosymbionts in biological associations, and (4), forming heterocysts (non-photosynthetic, O 2-free cells) in some filamentous taxa, which allows N 2 fixation to proceed while receiving photo-reductant and organic C through photosynthesis from adjacent cells [8].

These are all remarkably clever adaptations to a modern-day oxic biosphere, which help circumvent the “O 2 problem” [6]. From an ecosystem perspective, they have allowed N 2-fixing species to provide biologically available N from the vast reservoir of atmospheric N 2. However, on the ecosystem scale, recent N budget analyses indicate that N 2 fixation inputs fall far short of meeting ecosystem requirements when biologically available N inputs (from terrestrial and atmospheric sources) and losses (via denitrification, sedimentation and burial, and advection) are considered [9– 11]. As a result, freshwater, n class="Chemical">estuarine, anpan>d marine systems are oftenpan> pan> class="Disease">chronically N deficient [11– 17]. Pervasive N limitation has many implications for ecosystem function, especially when excessive external nutrient inputs lead to accelerating primary production (eutrophication), harmful algal blooms, and excessive O 2 consumption (hypoxia). If chronic N-limited conditions prevail in water bodies and N 2 fixation cannot meet ecosystem N requirements, then external N inputs often supply N to support eutrophication and its unwanted symptoms. From a management perspective, this means that the growing global glut of N inputs from agricultural, urban, and industrial sources [14, 18– 20] needs to be controlled, in addition to the broadly accepted phosphorus (P) input constraints, in order to protect our waterways and water supplies.

Why does N 2 fixation fall short of meeting ecosystem demands? Apparently, this process does not operate at sufficient rates in a modern-day, oxic world to compensate for losses via burial, export, and denitrification, even though it is protected and optimized by the various biological adaptations mentioned above. It is counteracted at larger scales by biogeochemical processes, such as denitrification, that run in the opposite direction (NO 3 → N 2). The N 2-fixing process is an energy-demanding one, requiring 16 n class="Chemical">ATP molecules to fix onpan>e molecule of N 2 [3]. In cyanpan>obacteria, this enpan>ergy demanpan>d has to be met by photosynpan>thesis, while in nonpan>-photosynpan>thetic bacteria, organpan>ic matter anpan>d redox reactionpan>s serve as enpan>ergy sources [3]. In highly productive (eutrophic), turbid waters where cyanpan>obacteria anpan>d bacteria thrive, the availability of photosynpan>thetically active radiationpan> (PAR: 400–700 nm) is oftenpan> restricted, causing a radianpan>t enpan>ergy deficit anpan>d suboptimal N 2 fixationpan> rates. Seconpan>dly, cyanpan>obacteria taxa that dominate in eutrophic waters oftenpan> accumulate as thick surface “blooms”, in part to circumvenpan>t light limitationpan> in subsurface waters [11]. High rates of photosynpan>thesis in such blooms lead to O 2 supersaturationpan>, oftenpan> in excess of 200% saturationpan> [21]. These ambienpan>t O 2 levels inhibit N 2 fixationpan> in situ, evenpan> in heterocystous taxa [22, 23]. Thirdly, N 2 fixationpan> requires high levels of P (to support the enpan>ergetics, e.g. pan> class="Chemical">ATP formation and nucleic acid production) and metals, most prominently iron (Fe), which is a co-factor in the enzyme complex nitrogenase [3]. In highly oxygenated surface waters, Fe occurs as the insoluble and biologically unavailable Fe 3+ ion that may lead to Fe-limited conditions [24]. Lastly, wind-induced turbulence and vertical mixing can reduce N 2 fixation potential by disrupting colonies and aggregates and enhancing inward diffusion of O 2 ( Figure 1) [25] and deepening the mixed layer, reducing light availability.

Figure 1.

The nitrogen fixing process, as mediated by cyanobacteria (utilizing oxygenic photosynthesis as an energy and carbon source) as well as heterotrophic and chemolithotrophic microorganisms, in eutrophic surface waters.

Potential environmental controls, including phosphorus (P) and iron (Fe) availability, energy sources, and dissolved oxygen inhibition, are shown in red. The background photo is of an O 2-supersaturated (during daytime) cyanobacterial surface bloom in Lake Taihu, China. Photograph by H. Paerl.

Potential envn class="Chemical">ironmenpan>tal conpan>trols, including pan> class="Chemical">phosphorus (P) and iron (Fe) availability, energy sources, and dissolved oxygen inhibition, are shown in red. The background photo is of an O 2-supersaturated (during daytime) cyanobacterial surface bloom in Lake Taihu, China. Photograph by H. Paerl.

Thus, while N 2 fixation converts inert N 2 into biologically available NH 3 to support aquatic n class="Chemical">fertility in a remarkable fashionpan>, it faces multiple conpan>straints anpan>d limitationpan>s in aquatic enpan>vironments, especially in surface waters, which are often N limited. Geochemists, some limnologists, and a few oceanographers have assumed that as long as P and Fe are readily available, N 2 fixation should make up for an N deficit, given the unlimited supply of N 2 available [26, 27]. However, this assumed linear stoichiometric relationship is not straightforward. Major environmental factors constrain this process, preventing it from functioning at optimal rates and supplying complete ecosystem N requirements [8, 11]. As a result, much of the world’s marine and freshwater environments remain chronically N deficient. In practical (management) terms, this limitation means that external inputs of N play a key role in providing adequate and excessive fertility (eutrophication) of many freshwater and most marine ecosystems [11, 15, 16]. Tremendous increases in anthropogenically generated bioavailable N in the form of synthetic (Haber process) fertilizers, agricultural, industrial, and urban wastes, and N 2 emissions (as both oxides and reduced forms of N) far overshadow biological fixation of N 2 in providing available N to receiving waters. Effective future management and protection of our fresh and marine waters will depend on the control of external inputs of both N and P [11, 27] instead of depending on the more traditional approach of controlling P inputs without N restrictions [28].