The surface water of the marine environment has traditionally been viewed as a nitrogen (N) limited habitat, and this has guided the development of conceptual biogeochemical models focusing largely on the reservoir of nitrate as the critical source of N to sustain primary productivity. However, selected groups of Bacteria, including cyanobacteria, and Archaea can utilize dinitrogen (N2) as an alternative N source. In the marine environment, these microorganisms can have profound effects on net community production processes and can impact the coupling of C-N-P cycles as well as the net oceanic sequestration of atmospheric carbon dioxide. As one component of an integrated ‘Nitrogen Transport and Transformations’ project, we have begun to re-assess our understanding of (1) the biotic sources and rates of N2 fixation in the world’s oceans, (2) the major controls on rates of oceanic N2 fixation, (3) the significance of this N2 fixation for the global carbon cycle and (4) the role of human activities in the alteration of oceanic N2 fixation. Preliminary results indicate that rates of N2 fixation, especially in subtropical and tropical open ocean habitats, have a major role in the global marine N budget. Iron (Fe) bioavailability appears to be an important control and is, therefore, critical in extrapolation to global rates of N2 fixation. Anthropogenic perturbations may alter N2 fixation in coastal environments through habitat destruction and eutrophication, and open ocean N2 fixation may be enhanced by warming and increased stratification of the upper water column. Global anthropogenic and climatic changes may also affect N2 fixation rates, for example by altering dust inputs (i.e. Fe) or by expansion of subtropical boundaries. Some recent estimates of global ocean N2 fixation are in the range of 100−200 Tg N (1−2 × 1014 g N) yr −1, but have large uncertainties. These estimates are nearly an order of magnitude greater than historical, pre-1980 estimates, but approach modern estimates of oceanic denitrification.

Dinitrogen fixation in the world's oceans

Molybdenum and uranium geochemistry in continental margin sediments: Paleoproxy potential

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing. Images

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

The ratio of phosphate and nitrate concentrations in the deep ocean matches closely the Redfield ratio required by phytoplankton growing in the surface ocean. Furthermore, the oxygen available from dissolution in ocean water is, on average, just sufficient for the respiration of the resulting organic matter. We review various feedback mechanisms that have been proposed to account for these remarkable correspondences and construct a model to test their effectiveness. The model's initial steady state is cate responds to perturbation in 1000–2000 years and phosphate in 40,000-60,000 years. However, recently increased estimates oflose to the Redfield ratios and stable against instantaneous changes in the sizes of the nitrate and phosphate reservoirs. When classic flux estimates are adopted, nitr the input and output fluxes of nitrate and phosphate suggest that they respond more rapidly to perturbation, nitrate in 500–1000 years and phosphate in 10,000–15,000 years. Nitrogen fixation tends to maintain nitrate close to Redfield ratio with phosphate, while denitrification tends to keep nitrate as the proximate limiting nutrient and tie it in Redfield ratio to dissolved oxygen. Under increases in phosphorus input to the ocean, the relative responsiveness of nitrogen fixation and denitrification determine whether nitrate remains close to Redfield ratio to phosphate or to oxygen. If nitrogen fixation is strongly limited (e.g., by lack of iron), increasing phosphorus input to the ocean can cause phosphate to deviate above Redfield ratio to nitrate. Hence nitrogen dynamics can control phosphate behavior and nitrate can potentially be the ultimate limiting nutrient over geologic periods of time. When nitrate and phosphate are coupled together by responsive nitrogen fixation, negative feedbacks on organic and calcium-bound phosphorus burial stabilize their concentrations. If anoxia suppresses organic phosphorus burial, the resulting feedbacks on phosphate (positive) and oxygen (negative) improve regulation toward the Redfield ratios. Variants of the model are forced with a global record of phosphorus accumulation in biogenic sediments as a proxy for changes in phosphate input to the ocean over the past 40 Myr. Nitrate is generally regulated close to Redfield ratio to phosphate, despite large changes in phosphorus input. If nitrogen fixation is strongly limited, then there is one interval (∼15 Myr ago) when a very rapid increase in phosphate input forces phosphate above Redfield ratio to nitrate. Decreases in phosphorus input cause phosphate and nitrate to quickly deviate below Redfield ratio with oxygen, removing anoxia from the ocean, while increases in phosphorus input rapidly increase anoxia. Hence we conclude that there appears to be an element of chance in observing today's ocean “on the edge of anoxia” with nitrate, phosphate, and oxygen all close to the Redfield ratios.

https://agupubs.onlinelibrary.wiley.com/doi/pdf/10.1029/1999GB900065

Redfield revisited: 1. Regulation of nitrate, phosphate, and oxygen in the ocean

Cyanobacteria (blue-green algae) in the genera Anabaena, Aphanizomenon, Microcystis, and Oscillatoria often form extensive and persistent blooms in freshwater aquaculture ponds. Bloom-forming cyanobacteria are undesirable in aquaculture ponds because: 1) they are a relatively poor base for aquatic food chains; 2) they are poor oxygenators of the water and have undesirable growth habits; 3) some species produce odorous metabolites that impart undesirable flavors to the cultured animal; and 4) some species may produce compounds that are toxic to aquatic animals. Development of cyanobacterial blooms is favored under conditions of high nutrient loading rates (particularly if the availability of nitrogen is limited relative to phosphorus), low rates of vertical mixing, and warm water temperatures. Under those conditions, dominance of phytoplankton communities by cyanobacteria is the result of certain unique physiological attributes (in particular, N2 fixation and buoyancy regulation) that allow cyanobacteria to compete effectively with other phytoplankton. The ability to fix N2 provides a competitive advantage under severe nitrogen limitation because it allows certain cyanobacterial species to make use of a source of nitrogen unavailable to other phytoplankton. The ability to regulate cell buoyancy through environmentally-controlled collapse ad reformation of intracellular gas vacuoles is perhaps the primary reason for the frequent dominance of aquaculture pond phytoplankton communities by cyanobacteria. Cyanobacteria that can regulate their position in the water column gain a distinct advantage over other phototrophs in poorly mixed bodies of water. In addition to the physicochemical interactions that influence phytoplankton community dynamics, cyanobacterial-microbial associations may play an important regulatory role in determining community structure. Cyanobacteria are always found in close association with a diverse array of microorganisms, including eubacteria, fungi, and protozoans. These associations, which in the past have often been viewed as antagonistic, are increasingly seen as mutualistic and may function in a positive manner during bloom development.

Ecology of Blue-Green Algae in Aquaculture Ponds

Nitrogen availability frequently controls phytoplankton growth in marine environments. It would seem that N,-fixing microorganisms would have a selective advantage in these environments, but N, fixation appears to be limited in most marine waters. Previous studies have focused on deficiencies of inorganic nutrients such as P, Fe, and especially MO as constraints on N, fixation. It was proposed that a high SOd2- : MoOd2- ratio interferes with cellular MoOd2- uptake and in turn results in insufficient quantities for incorporation into the N,-reducing enzyme complex nitrogenase. In the present study, in situ bioassays demonstrate that MO is not the limiting nutrient for N2 fixation by marine assemblages. Instead, enrichments of glucose or mannitol were found to initiate or increase NZ fixation in these assemblages. We also show that diazotrophic growth rates of an Azotobacter vinelandii strain containing a single MO-dependent nitrogenase are not inhibited at SOd2-- : MoOd2- ratios up to 8 times higher than those calculated for seawater. From these results we propose that widespread limitation of N, fixation in marine environments is due to deficiencies in organic matter and appropriate microhabitats rather than Mo.

https://aslopubs.onlinelibrary.wiley.com/doi/pdf/10.4319/lo.1991.36.7.1325

Evidence that molybdenum‐dependent nitrogen fixation is not limited by high sulfate concentrations in marine environments

Evidence for Two Dinitrogenase Reductases under Regulatory Control by Molybdenum in Azotobacter Vinelandii

14 – three genetically distinct nitrogenase systems in azotobacter vinelandii

Eight ineffective mutant strains were isolated from N-methyl-N'-nitro-N-nitrosoguanidine mutagenized cultures of cowpea Rhizobium strain 32H1. Strains CR1, CR2, CR3, CR4, CR5 and CR6 induced more, but smaller, nodules than the wild type. With the exception of strain CR2, these mutant strains reduced less than 1% of the amount of acetylene reduced by the wild type, in both the free-living and symbiotic assays. Strain CR2 reduced acetylene in the free-living assay but not in the symbiotic assay. Strains CR7 and CR8 responded variably (5–20% of the wild type) in free-living and symbiotic acetylene reduction assays. Nodules also varied from small white to normal-sized pink nodules. The phenotypic characteristics of the mutant strains were consistant with all leguminous plants tested and were stable upon reisolation from nodules. Fully effective revertants were selected from 4 of the ineffective mutant strains by the use of the leguminous plant,Macroptilium lathyroides. Serology, patterns of resistance to anti-bacterial agents, phage-typing, and antibiotic resistance markers were used to confirm strain identification.

Paul E. Bishop

Papers

Evidence that molybdenum‐dependent nitrogen fixation is not limited by high sulfate concentrations in marine environments

Evidence for Two Dinitrogenase Reductases under Regulatory Control by Molybdenum in Azotobacter Vinelandii

14 – three genetically distinct nitrogenase systems in azotobacter vinelandii

Isolation and partial characterization of fix− mutants from Rhizobium strain 32H1