Joseph P. Montoya

High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific ocean

The availability of nitrogen is important in regulating biological productivity in marine environments. Deepwater nitrate has long been considered the major source of new nitrogen supporting primary production in oligotrophic regions of the open ocean, but recent studies have showed that biological N2 fixation has a critical role in supporting oceanic new production. Large colonial cyanobacteria in the genus Trichodesmium and the heterocystous endosymbiont Richelia have traditionally been considered the dominant marine N2 fixers, but unicellular diazotrophic cyanobacteria and bacterioplankton have recently been found in the picoplankton and nanoplankton community of the North Pacific central gyre, and a variety of molecular and isotopic evidence suggests that these unicells could make a major contribution to the oceanic N budget. Here we report rates of N2 fixation by these small, previously overlooked diazotrophs that, although spatially variable, can equal or exceed the rate of N2 fixation reported for larger, more obvious organisms. Direct measurements of 15N2 fixation by small diazotrophs in various parts of the Pacific Ocean, including the waters off Hawaii where the unicellular diazotrophs were first characterized, show that N2 fixation by unicellular diazotrophs can support a significant fraction of total new production in oligotrophic waters.

Unicellular Cyanobacterial Distributions Broaden the Oceanic N2 Fixation Domain

Oceanic Nitrogen Fixation Nitrogen fixation in the oceans is important in sustaining global marine productivity and balances carbon dioxide export to the deep ocean. It was previously believed that marine nitrogen fixation was due to a single genus of filamentous cyanobacteria, Trichodesmium. The recent discovery of unicellular open-ocean cyanobacteria has raised the question of how they contribute to global ocean nitrogen fixation and how they compare in distribution and activity to Trichodesmium. Using data collected from the southwest Pacific Ocean, Moisander et al. (p. 1512, published online 25 February) show that the unicellular nitrogen-fixing cyanobacteria (UCYN-A and Crocosphaera watsonii) have distinct ecophysiologies and distinct oceanic distributions from each other, and from Trichodesmium. These data can be incorporated into models to retune estimates of the global rates of oceanic nitrogen fixation and carbon sequestration. Nitrogen fixation in the South Pacific Ocean is partitioned among several microbe species with distinct ecophysiologies. Nitrogen (N2)–fixing microorganisms (diazotrophs) are an important source of biologically available fixed N in terrestrial and aquatic ecosystems and control the productivity of oligotrophic ocean ecosystems. We found that two major groups of unicellular N2-fixing cyanobacteria (UCYN) have distinct spatial distributions that differ from those of Trichodesmium, the N2-fixing cyanobacterium previously considered to be the most important contributor to open-ocean N2 fixation. The distributions and activity of the two UCYN groups were separated as a function of depth, temperature, and water column density structure along an 8000-kilometer transect in the South Pacific Ocean. UCYN group A can be found at high abundances at substantially higher latitudes and deeper in subsurface ocean waters than Trichodesmium. These findings have implications for the geographic extent and magnitude of basin-scale oceanic N2 fixation rates.

Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean

The fresh water discharged by large rivers such as the Amazon is transported hundreds to thousands of kilometers away from the coast by surface plumes. The nutrients delivered by these river plumes contribute to enhanced primary production in the ocean, and the sinking flux of this new production results in carbon sequestration. Here, we report that the Amazon River plume supports N2 fixation far from the mouth and provides important pathways for sequestration of atmospheric CO2 in the western tropical North Atlantic (WTNA). We calculate that the sinking of carbon fixed by diazotrophs in the plume sequesters 1.7 Tmol of C annually, in addition to the sequestration of 0.6 Tmol of C yr−1 of the new production supported by NO3 delivered by the river. These processes revise our current understanding that the tropical North Atlantic is a source of 2.5 Tmol of C to the atmosphere [Mikaloff-Fletcher SE, et al. (2007) Inverse estimates of the oceanic sources and sinks of natural CO2 and the implied oceanic carbon transport. Global Biogeochem Cycles 21, doi:10.1029/2006GB002751]. The enhancement of N2 fixation and consequent C sequestration by tropical rivers appears to be a global phenomenon that is likely to be influenced by anthropogenic activity and climate change.

INTERACTIONS BETWEEN NITRATE UPTAKE AND NITROGEN FIXATION IN CONTINUOUS CULTURES OF THE MARINE DIAZOTROPH TRICHODESMIUM (CYANOBACTERIA)1

Diazotrophic cyanobacteria can take up combined nitrogen (nitrate, ammonium, amino acids, dissolved organic nitrogen) from solution, but the interaction between N2 fixation and uptake of combined nitrogen is not well understood. We studied the effects of combined nitrogen ) additions on N2 fixation rates in the cyanobacterium Trichodesmium erythraeum (IMS‐101) maintained in continuous culture in an N‐free medium (YBCII) and a 12:12‐h light:dark cycle. We measured acetylene reduction rates, nutrient concentrations, and biomass throughout the 12 h of illumination after the addition of nitrate (0.5–20 μM) at the start of the light period. Compared with unamended controls, Trichodesmium showed strong inhibition of acetylene reduction (up to 70%) in the presence of , with apparent saturation of the inhibition effect at an initial concentration of approximately 10 μM. The inhibition of acetylene reduction persisted through much of the light period as concentration in the culture vessel decreased. Recovery of N2 fixation was observed late in the light period in cultures amended with low concentrations of (<5 μM) when ambient concentrations had decreased to 0.3–0.4 μM in the culture vessel. Nitrate uptake accounted for as much as 86% of total N uptake and, at the higher treatment concentrations, more than made up for the observed decrease in N2 fixation rates. We conclude that Trichodesmium can obtain significant quantities of N through uptake of nitrate and does so in preference to N2 fixation when sufficient is available.

Turnover of carbon and nitrogen during growth of larval krill, Euphausia superba Dana: a stable isotope approach

Abstract Using natural abundances of stable isotopes (δ13C and δ15N) as tracers, carbon and nitrogen turnover rates were determined for larval krill, Euphausia superba Dana, maintained in the laboratory. Experimental populations of larvae were reared at +1.5°C and −1.5°C on foods of known isotopic composition and subsampled weekly (8–10 weeks) for a determination of wet weight and isotopic composition. Metabolic turnover of carbon and nitrogen, manifested as temporal shifts in δ13C and δ15N, was tied closely to temperature. Larval krill reared at +1.5°C had replaced 22–29% of their original body carbon at the conclusion of the experiment, but only 13–22% of their original body nitrogen. Larvae reared at −1.5°C exhibited little evidence of carbon turnover and replaced less than 6% of their original body nitrogen. These are the first simultaneous, coupled measurements of long-term carbon and nitrogen turnover for any marine animal, and provide an essential calibration for the interpretation of stable isotope ratios in animals collected from the field. In addition to the feeding experiments, animals were starved for 2 months at +1.5°C and −1.5°C. Starved krill exhibited little isotopic change. This finding suggests that starvation cannot account for large temporal variations observed in the isotopic composition of larval krill collected from the field.

Simulating the global distribution of nitrogen isotopes in the ocean

9 isotopes, 14 N and 15 N, in the nitrate (NO3 ), phytoplankton, zooplankton, and detritus 10 variables of the marine ecosystem model. The isotope effects of algal NO3 uptake, 11 nitrogen fixation, water column denitrification, and zooplankton excretion are considered 12 as well as the removal of NO3 by sedimentary denitrification. A global database of 13 d 15 NO3 observations is compiled from previous studies and compared to the model 14 results on a regional basis where sufficient observations exist. The model is able to 15 qualitatively and quantitatively reproduce many of the observed patterns such as high 16 subsurface values in water column denitrification zones and the meridional and vertical 17 gradients in the Southern Ocean. The observed pronounced subsurface minimum in the 18 Atlantic is underestimated by the model presumably owing to too little simulated 19 nitrogen fixation there. Sensitivity experiments reveal that algal NO3 uptake, nitrogen 20 fixation, and water column denitrification have the strongest effects on the simulated 21 distribution of nitrogen isotopes, whereas the effect from zooplankton excretion is 22 weaker. Both water column and sedimentary denitrification also have important indirect 23 effects on the nitrogen isotope distribution by reducing the fixed nitrogen inventory, 24 which creates an ecological niche for nitrogen fixers and, thus, stimulates additional N2 25 fixation in the model. Important model deficiencies are identified, and strategies for 26 future improvement and possibilities for model application are outlined.

Natural isotopic composition of dissolved inorganic nitrogen in the Chesapeake Bay

The natural abundances of 15N in the dissolved inorganic pools of nitrogen in the Chesapeake Bay were measured in the spring and fall of 1984. Changes in the δ15N of NH4+ and the combined pool of (NO3− + NO2−) reflected both seasonal and short-term changes in the estuarine nitrogen cycle. In the spring, oxidation of NH4+ at the head of the bay in the region of the turbidity maximum and in localized regions throughout the bay, led to elevated values of δ15N in the NH4+ pool. The δ15N of the (NO3− + NO2−) pool tended to increase toward the south, enabling an estimate of the isotopic fractionation factor for the consumption of NO3− to be derived; the estimate (1·0070), is similar to literature values of the fractionation factor for NO3− uptake by phytoplankton, supporting previous research suggesting that phytoplankton uptake is the major sink for NO3− in the bay. Denitrification led to elevated values of δ15N in the (NO3− + NO2−) pool in deep water. Over the course of the summer, the δ15N of NH4+ increased throughout the bay. A significant correlation was found between the δ15N of the NH4+ pool and the concentration of NO2− both above and below the pycnocline during the fall cruise, suggesting that the increase in the δ15N of the NH4+ pool was due to the oxidation of NH4+. In the fall, changes were also observed in the δ15N of both the NH4+ and (NO3− + NO2−) pools which were consistent with the occurrence of NH4+ oxidation. From these changes, a fractionation factor for NH4+ oxidation between 1·0120 and 1·0167 was derived, which is similar to values reported in the literature.

Relating low δ15N values of zooplankton to N2-fixation in the tropical North Atlantic: insights provided by stable isotope ratios of amino acids

Abstract Recent evaluations of the global nitrogen budget include greatly increased estimates of N2-fixation in oceanic waters. Low stable N isotope ratios in planktonic food webs of tropical and subtropical oceans have been used as one indication of the importance of N2-fixation. Interpretation of bulk stable N isotope ratios can, however, be confounded when the source and process information that they contain cannot be separated clearly. In this paper, we use stable N isotope ratios of amino acids to help separate source and trophic effects associated with changes in bulk stable N isotope ratios of zooplankton across the tropical North Atlantic. Patterns in stable N isotope ratios of amino acids along a transect from the Cape Verde Islands to Barbados identify a change in N source supporting zooplankton production, and virtually no change in the trophic position of zooplankton size classes from the eastern to the western side of the tropical North Atlantic. Furthermore, comparison of stable N isotope ratios of amino acids in zooplankton with those in Trichodesmium suggests that diazotrophs are the source of the low stable N isotope ratios at the western end of the transect. The evidence provided by stable N isotope ratios of amino acids supports the interpretation of large-scale patterns in bulk stable N isotope ratios that N2-fixation indeed makes a major contribution to the global N budget.

Abundance and distribution of major groups of diazotrophic cyanobacteria and their potential contribution to N2 fixation in the tropical Atlantic Ocean.

The abundances of six N₂-fixing cyanobacterial phylotypes were profiled at 22 stations across the tropical Atlantic Ocean during June 2006, and used to model the contribution of the diazotrophs to N₂ fixation. Diazotroph abundances were measured by targeting the nifH gene of Trichodesmium, unicellular groups A, B, C (UCYN-A, UCYN-B and UCYN-C), and diatom-cyanobiont symbioses Hemiaulus-Richelia, Rhizosolenia-Richelia and Chaetoceros-Calothrix. West to east gradients in temperature, salinity and nutrients [NO₃⁻ + NO₂⁻, PO₄³⁻, Si(OH)₄] showed the influence of the Amazon River plume and its effect on the distributions of the diazotrophs. Trichodesmium accounted for more than 93% of all nifH genes detected, dominated the warmer waters of the western Atlantic, and was the only diazotroph detected at the equatorial upwelling station. UCYN-A was the next most abundant (> 5% of all nifH genes) and dominated the cooler waters of the eastern Atlantic near the Cape Verde Islands. UCYN-C was found at a single depth (200 m) of high salinity and low temperature and nutrients, whereas UCYN-B cells were widespread but in very low abundance (6.1 × 10¹ ± 4.6 × 10² gene copies l⁻¹). The diatom-cyanobionts were observed primarily in the western Atlantic within or near the high Si(OH)₄ input of the Amazon River plume. Overall, highest diazotroph abundances were observed at the surface and declined with depth, except for some subsurface peaks in Trichodesmium, UCYN-B and UCYN-A. Modelled contributions of Trichodesmium, UCYN-B and UCYN-A to total N₂ fixation suggested that Trichodesmium had the largest input, except for the potential of UCYN-A at the Cape Verde Islands.

Microbial community gene expression within colonies of the diazotroph, Trichodesmium, from the Southwest Pacific Ocean.

Trichodesmium are responsible for a large fraction of open ocean nitrogen fixation, and are often found in complex consortia of other microorganisms, including viruses, prokaryotes, microbial eukaryotes and metazoa. We applied a community gene expression (metatranscriptomic) approach to study the patterns of microbial gene utilization within colonies of Trichodesmium collected during a bloom in the Southwest Pacific Ocean in April 2007. The survey generated 5711-day and 5385-night putative mRNA reads. The majority of mRNAs were from the co-occurring microorganisms and not Trichodesmium, including other cyanobacteria, heterotrophic bacteria, eukaryotes and phage. Most transcripts did not share homology with proteins from cultivated microorganisms, but were similar to shotgun sequences and unannotated proteins from open ocean metagenomic surveys. Trichodesmium transcripts were mostly expressed photosynthesis, N2 fixation and S-metabolism genes, whereas those in the co-occurring microorganisms were mostly involved in genetic information storage and processing. Detection of Trichodesmium genes involved in P uptake and As detoxification suggest that local enrichment of N through N2 fixation may lead to a P-stress response. Although containing similar dominant transcripts to open ocean metatranscriptomes, the overall pattern of gene expression in Trichodesmium colonies was distinct from free-living pelagic assemblages. The identifiable genes expressed by Trichodesmium and closely associated microorganisms reflect the constraints of life in well-lit and nutrient-poor waters, with biosynthetic investment in nutrient acquisition and cell maintenance, which is in contrast to gene transcription by soil and coastal seawater microbial assemblages. The results provide insight into aggregate microbial communities in contrast to planktonic free-living assemblages that are the focus of other studies.