Symbioses between nitrogen (N)(2)-fixing prokaryotes and photosynthetic eukaryotes are important for nitrogen acquisition in N-limited environments. Recently, a widely distributed planktonic uncultured nitrogen-fixing cyanobacterium (UCYN-A) was found to have unprecedented genome reduction, including the lack of oxygen-evolving photosystem II and the tricarboxylic acid cycle, which suggested partnership in a symbiosis. We showed that UCYN-A has a symbiotic association with a unicellular prymnesiophyte, closely related to calcifying taxa present in the fossil record. The partnership is mutualistic, because the prymnesiophyte receives fixed N in exchange for transferring fixed carbon to UCYN-A. This unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N(2) fixation.

Abstract. Marine N2 fixing microorganisms, termed diazotrophs, are a key functional group in marine pelagic ecosystems. The biological fixation of dinitrogen (N2) to bioavailable nitrogen provides an important new source of nitrogen for pelagic marine ecosystems and influences primary productivity and organic matter export to the deep ocean. As one of a series of efforts to collect biomass and rates specific to different phytoplankton functional groups, we have constructed a database on diazotrophic organisms in the global pelagic upper ocean by compiling about 12 000 direct field measurements of cyanobacterial diazotroph abundances (based on microscopic cell counts or qPCR assays targeting the nifH genes) and N2 fixation rates. Biomass conversion factors are estimated based on cell sizes to convert abundance data to diazotrophic biomass. The database is limited spatially, lacking large regions of the ocean especially in the Indian Ocean. The data are approximately log-normal distributed, and large variances exist in most sub-databases with non-zero values differing 5 to 8 orders of magnitude. Reporting the geometric mean and the range of one geometric standard error below and above the geometric mean, the pelagic N2 fixation rate in the global ocean is estimated to be 62 (52–73) Tg N yr−1 and the pelagic diazotrophic biomass in the global ocean is estimated to be 2.1 (1.4–3.1) Tg C from cell counts and to 89 (43–150) Tg C from nifH-based abundances. Reporting the arithmetic mean and one standard error instead, these three global estimates are 140 ± 9.2 Tg N yr−1, 18 ± 1.8 Tg C and 590 ± 70 Tg C, respectively. Uncertainties related to biomass conversion factors can change the estimate of geometric mean pelagic diazotrophic biomass in the global ocean by about ±70%. It was recently established that the most commonly applied method used to measure N2 fixation has underestimated the true rates. As a result, one can expect that future rate measurements will shift the mean N2 fixation rate upward and may result in significantly higher estimates for the global N2 fixation. The evolving database can nevertheless be used to study spatial and temporal distributions and variations of marine N2 fixation, to validate geochemical estimates and to parameterize and validate biogeochemical models, keeping in mind that future rate measurements may rise in the future. The database is stored in PANGAEA (doi:10.1594/PANGAEA.774851).

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N2-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N2 fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N2 fixation (1.15–71.5 fmol N per cell h−1) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N2 fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N2 fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.

Nitrogen (N(2))-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N(2)-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.

The vertical and horizontal distributions of seven diazotrophic populations in the western tropical north Atlantic (WTNA) Ocean were examined using a nifH DNA quantitative polymerase chain reaction (QPCR) approach. The nifH phylotype abundances were highest near the surface and decreased with depth, with the exception of the cyanobacterial symbiont Calothrix, which was not detected at any station. Richelia associated with the diatoms Rhizosolenia clevei and Hemiaulus hauckii were distributed within the freshwater lens of the Amazon plume. Abundances of H. hauckii‐Richelia nifH genes dominated all depths in 6 of 10 vertical profiles and 10 of 20 surface samples. In addition, estimates of Richelia associated with H. hauckii increased northwest (8‐ 12°N, 56‐54°W) from the river mouth, where significantly ( p < 0.001) higher abundances (>105 copies L−1) were found in mesohaline waters (31‐34.9). nifH copy abundance for surface populations of the H. hauckii‐Richelia symbioses were positively correlated (r2 = 0.59) with salinity. Three unicellular cyanobacterial groups and Trichodesmium had similar horizontal distributions, where the highest nifH copy estimates were at stations with salinity ≥35 and northeast (6‐10°N 50°W) of the freshwater lens. The abundance of Trichodesmium spp. and unicellular Group B nifH gene copies co‐varied (r2 = 0.60). The QPCR study showed the dominance of H. hauckii‐Richelia symbioses in the Amazon plume waters, implying that these associations had an ecological advantage over the other diazotrophs. Outside of the plume nutrients were below detection, abundances of freeliving unicellular cyanobacterial phylotypes, including a novel group designated Group C, were abundant (>105 copies L−1) and comparable to the abundances of Trichodesmium spp. Thus, there appeared to be a cascade of diazotrophic communities along gradients of salinity and nutrients in the WTNA.

An Advanced Laser Fluorometer (ALF) capable of discriminating several phytoplankton pigment types was utilized in conjunction with microscopic data to map the distribution of phytoplankton communities in the Amazon River plume in May-June-2010, when discharge from the river was at its peak. Cluster analysis and Non-metric Multi-Dimensional Scaling (NMDS) helped distinguish three distinct biological communities that separated largely on the basis of salinity gradients across the plume. These three communities included an ''estuarine type'' comprised of a high biomass mixed population of diatoms, cryptophytes and green-water Synechococcus spp. located upstream of the plume, a ''mesohaline type'' made up largely of communities of Diatom-Diazotroph Associations (DDAs) and located in the northwestern region of the plume and an ''oceanic type'' in the oligotrophic waters outside of the plume made up of Trichodesmium and Synechococcus spp. Although salinity appeared to have a substantial influence on the distribution of different phytoplankton groups, ALF and microscopic measurements examined in the context of the hydro-chemical environment of the river plume, helped establish that the phytoplankton community structure and distribution were strongly controlled by inorganic nitrate plus nitrite (NO 3 + NO 2) availability whose concentrations were low throughout the plume. Towards the southern, low-salinity region of the plume, NO 3 + NO 2 supplied by the onshore flow of subsurface ($80 m depth) water, ensured the continuous sustenance of the mixed phytoplankton bloom. The large drawdown of SiO 3 and PO 4 associated with this ''estuarine type'' mixed bloom at a magnitude comparable to that observed for DDAs in the mesohaline waters, leads us to contend that, diatoms, cryptophytes and Synechococcus spp., fueled by the offshore influx of nutrients also play an important role in the cycling of nutrients in the Amazon River plume.

Investigating the contribution of microbial populations to biochemical processes of global significance is challenging as there are few approaches that can detect microbial metabolic activities on single-cell level. Given the widespread distribution and importance of microorganisms in elemental transformations, improved methods for measuring microbial activities in naturally occurring microbial communities is essential. In this article, microautoradiography (MAR), Raman microspectroscopy, and Secondary Ion Mass Spectrometry (SIMS) and their combination with isotope labeling and molecular genetic methods for cell identification (i.e. FISH and related methods) are reviewed. We focus our review on the application of MAR-FISH, Raman-FISH, and FISH-SIMS to environmental samples, with a more detailed description of the use of nanoSIMS-based methodologies to identify, quantify, and visualize the incorporation of labeled substrates of single microorganisms in complex microbial communities. We highlight examples from the marine habitat. In addition, relevant technical aspects as well as important considerations concerning sample preparation and handling are presented. We conclude with a perspective on the usefulness of such tools to study the role of microorganisms in biogeochemical cycling from micron to global scales.

N 2 fixation has been understudied in marine environments outside of the subtropical and tropical oceans and where water temperatures are typically below 20-25uC. We identified nifH phylotypes and measured N 2 fixation rates under ambient conditions (maximum of 19uC) in water collected 750 km off the coast of California in oligotrophic waters of the North Pacific Ocean (34uN, 129uW). Near-surface N 2 fixation rates averaged 0.25 6 0.05 nmol N L 21 d 21 for 24 incubation bottles. Despite low ambient concentrations of iron (,0.1 nmol L 21 ) and phosphorus (,0.3 mmol L 21 ), N 2 fixation rates were unaffected by iron and phosphorus amendments. Using reverse transcription-quantitative polymerase chain reaction (RT-QPCR) methodology, we estimated transcript abundance and patterns of expression for several unicellular diazotrophs, including the group A phylotype, which showed the highest daily mRNA abundances. The N 2 -fixing assemblage extended to 60-80 m depth, well below the seasonal thermocline (40 m). The calculated areal N 2 fixation rate (15 mmol N m 22 d 21 ) was small compared with estimates from other regions of the Pacific; however, the estimated fixation rate was similar to other published results, suggesting that processes other than cellular growth rate may determine the abundance of unicellular diazotrophs. Despite the low N 2 fixation rates, the new nitrogen added to the euphotic zone by N 2 fixation could account for at least 10% of new production during the study period.