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Biological nitrogen (N 2 ) fixation is critical in global biogeochemical cycles and in sustaining the productivity of the oceans. There remain many unanswered questions, unresolved hypotheses, and unchallenged paradigms. The fundamental balance of N input and losses has not been fully resolved. One of the major N 2 -fixers, Trichodesmium, remains an enigma with intriguing biological and ecological secrets. Cyanobacterial N 2 fixation, once thought to be primarily due to free-living cyanobacteria, now also appears to be dependent on microbial interactions, from microbiomes to unicellular symbioses, which remain poorly characterized. Nitrogenase genes associated with diverse non-cyanobacterial diazotrophs (NCDs) are prevalent, but their significance remains a huge knowledge gap. Answering questions, new and old, such as those discussed here, is needed to understand the ocean's N and C cycles and their responses to environmental change., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Trichodesmium is an important dinitrogen (N 2 )-fixing cyanobacterium in marine ecosystems. Recent nucleic acid analyses indicate that Trichodesmium colonies with their diverse epibionts support various nitrogen (N) transformations beyond N 2 fixation. However, rates of these transformations and concentration gradients of N compounds in Trichodesmium colonies remain largely unresolved. We combined isotope-tracer incubations, micro-profiling and numeric modelling to explore carbon fixation, N cycling processes as well as oxygen, ammonium and nitrate concentration gradients in individual field-sampled Trichodesmium colonies. Colonies were net-autotrophic, with carbon and N 2 fixation occurring mostly during the day. Ten percent of the fixed N was released as ammonium after 12-h incubations. Nitrification was not detectable but nitrate consumption was high when nitrate was added. The consumed nitrate was partly reduced to ammonium, while denitrification was insignificant. Thus, the potential N transformation network was characterised by fixed N gain and recycling processes rather than denitrification. Oxygen concentrations within colonies were ~60-200% air-saturation. Moreover, our modelling predicted steep concentration gradients, with up to 6-fold higher ammonium concentrations, and nitrate depletion in the colony centre compared to the ambient seawater. These gradients created a chemically heterogeneous microenvironment, presumably facilitating diverse microbial metabolisms in millimetre-sized Trichodesmium colonies.

The response of the prominent marine dinitrogen (N 2 )-fixing cyanobacteria Trichodesmium to ocean acidification (OA) is critical to understanding future oceanic biogeochemical cycles. Recent studies have reported conflicting findings on the effect of OA on growth and N 2 fixation of Trichodesmium. Here, we quantitatively analyzed experimental data on how Trichodesmium reallocated intracellular iron and energy among key cellular processes in response to OA, and integrated the findings to construct an optimality-based cellular model. The model results indicate that Trichodesmium growth rate decreases under OA primarily due to reduced nitrogenase efficiency. The downregulation of the carbon dioxide (CO 2 )-concentrating mechanism under OA has little impact on Trichodesmium, and the energy demand of anti-stress responses to OA has a moderate negative effect. We predict that if anthropogenic CO 2 emissions continue to rise, OA could reduce global N 2 fixation potential of Trichodesmium by 27% in this century, with the largest decrease in iron-limiting regions.

The N 2 -fixing cyanobacterium Trichodesmium is intensely studied because of the control this organism exerts over the cycling of carbon and nitrogen in the low nutrient ocean gyres. Although iron (Fe) and phosphorus (P) bioavailability are thought to be major drivers of Trichodesmium distributions and activities, identifying resource controls on Trichodesmium is challenging, as Fe and P are often organically complexed and their bioavailability to a single species in a mixed community is difficult to constrain. Further, Fe and P geochemistries are linked through the activities of metalloenzymes, such as the alkaline phosphatases (APs) PhoX and PhoA, which are used by microbes to access dissolved organic P (DOP). Here we identified significant correlations between Trichodesmium-specific transcriptional patterns in the North Atlantic (NASG) and North Pacific Subtropical Gyres (NPSG) and patterns in Fe and P biogeochemistry, with the relative enrichment of Fe stress markers in the NPSG, and P stress markers in the NASG. We also observed the differential enrichment of Fe-requiring PhoX transcripts in the NASG and Fe-insensitive PhoA transcripts in the NPSG, suggesting that metalloenzyme switching may be used to mitigate Fe limitation of DOP metabolism in Trichodesmium. This trait may underpin Trichodesmium success across disparate ecosystems.

Distribution of diazotrophs and their nitrogen fixation activity were investigated in the northern South China Sea (nSCS) and the Kuroshio from July 16th to September 1st, 2009. N 2 fixation activities in whole seawater and <10 μm fraction at the surface were measured by acetylene reduction assay. Higher activities were observed at the East China Sea (ECS) Kuroshio and the nSCS shelf. The nSCS basin showed a low N 2 fixation activity. The <10 μm fractions (unicellular diazotrophs) contributed major portion to the whole-water activity in the survey time, indicating that nanoplanktonic cyanobacterias were the major diazotrophs in the survey area. Daily N 2 fixation rates of Trichodesmium ranged from 0.11 to 9.83 pmolNtrichome -1 d -1 with an average of 4.03 pmolNtrichome -1 d -1 . The Luzon Strait and the ECS Kuroshio had higher N 2 fixation rates of Trichodesmium than the nSCS shelf and basin. Calculated activities of Trichodesmium at most stations were moderately low compared with that of the whole-water. The contribution of N 2 fixation by the whole-water to primary production ranged from 1.7% to 18.5%. The estimated amount of new nitrogen introduced by Trichodesmium contributed up to 0.14% of the total primary production and 0.41% of the new production in the Luzon Strait.

Nitrogen-fixing (N 2 ) cyanobacteria provide bioavailable nitrogen to vast ocean regions but are in turn limited by iron (Fe) and/or phosphorus (P), which may force them to employ alternative nitrogen acquisition strategies. The adaptive responses of nitrogen fixers to global-change drivers under nutrient-limited conditions could profoundly alter the current ocean nitrogen and carbon cycles. Here, we show that the globally important N 2 fixer Trichodesmium fundamentally shifts nitrogen metabolism toward organic-nitrogen scavenging following long-term high-CO 2 adaptation under iron and/or phosphorus (co)limitation. Global shifts in transcripts and proteins under high-CO 2 /Fe-limited and/or P-limited conditions include decreases in the N 2 -fixing nitrogenase enzyme, coupled with major increases in enzymes that oxidize trimethylamine (TMA). TMA is an abundant, biogeochemically important organic nitrogen compound that supports rapid Trichodesmium growth while inhibiting N 2 fixation. In a future high-CO 2 ocean, this whole-cell energetic reallocation toward organic nitrogen scavenging and away from N 2 fixation may reduce new-nitrogen inputs by Trichodesmium while simultaneously depleting the scarce fixed-nitrogen supplies of nitrogen-limited open-ocean ecosystems. IMPORTANCE is among the most biogeochemically significant microorganisms in the ocean, since it supplies up to 50% of the new nitrogen supporting open-ocean food webs. We used Trichodesmium is among the most biogeochemically significant microorganisms in the ocean, since it supplies up to 50% of the new nitrogen supporting open-ocean food webs. We used Trichodesmium cultures adapted to high-CO 2 and concurrent nutrient limitation(s) fundamentally shift nitrogen metabolism away from nitrogen fixation and instead toward upregulation of organic nitrogen-scavenging pathways. We show that the responses of 2 to projected future ocean conditions include decreases in the nitrogen-fixing nitrogenase enzymes coupled with major increases in enzymes that oxidize the abundant organic nitrogen source trimethylamine (TMA). Such a shift toward organic nitrogen uptake and away from nitrogen fixation may substantially reduce new-nitrogen inputs by Trichodesmium to projected future ocean conditions include decreases in the nitrogen-fixing nitrogenase enzymes coupled with major increases in enzymes that oxidize the abundant organic nitrogen source trimethylamine (TMA). Such a shift toward organic nitrogen uptake and away from nitrogen fixation may substantially reduce new-nitrogen inputs by Trichodesmium ocean, with potential global implications for ocean carbon and nitrogen cycling.2 ocean, with potential global implications for ocean carbon and nitrogen cycling., (Copyright © 2018 American Society for Microbiology.)

The diazotrophic cyanobacterium, Trichodesmium, is an integral component of the marine nitrogen cycle and contributes significant amounts of new nitrogen to oligotrophic, tropical/subtropical ocean surface waters. Trichodesmium forms macroscopic, fusiform (tufts), spherical (puffs) and raft-like colonies that provide a pseudobenthic habitat for a host of other organisms including marine invertebrates, microeukaryotes and numerous other microbes. The diversity and activity of denitrifying bacteria found in association with the colonies was interrogated using a series of molecular-based methodologies targeting the gene encoding the terminal step in the denitrification pathway, nitrous oxide reductase (nosZ). Trichodesmium spp. sampled from geographically isolated ocean provinces (the Atlantic Ocean, the Red Sea and the Indian Ocean) were shown to harbor highly similar, taxonomically related communities of denitrifiers whose members are affiliated with the Roseobacter clade within the Rhodobacteraceae (Alphaproteobacteria). These organisms were actively expressing nosZ in samples taken from the mid-Atlantic Ocean and Red Sea implying that Trichodesmium colonies are potential sites of nitrous oxide consumption and perhaps earlier steps in the denitrification pathway also. It is proposed that coupled nitrification of newly fixed N is the most likely source of nitrogen oxides supporting nitrous oxide cycling within Trichodesmium colonies., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Gradients of oxygen (O 2 ) and pH, as well as small-scale fluxes of carbon (C), nitrogen (N) and O 2 were investigated under different partial pressures of carbon dioxide (pCO 2 ) in field-collected colonies of the marine dinitrogen (N 2 )-fixing cyanobacterium Trichodesmium. Microsensor measurements indicated that cells within colonies experienced large fluctuations in O 2 , pH and CO 2 concentrations over a day-night cycle. O 2 concentrations varied with light intensity and time of day, yet colonies exposed to light were supersaturated with O 2 (up to ~200%) throughout the light period and anoxia was not detected. Alternating between light and dark conditions caused a variation in pH levels by on average 0.5 units (equivalent to 15 nmol l -1 proton concentration). Single-cell analyses of C and N assimilation using secondary ion mass spectrometry (SIMS; large geometry SIMS and nanoscale SIMS) revealed high variability in metabolic activity of single cells and trichomes of Trichodesmium, and indicated transfer of C and N to colony-associated non-photosynthetic bacteria. Neither O 2 fluxes nor C fixation by Trichodesmium were significantly influenced by short-term incubations under different pCO 2 levels, whereas N 2 fixation increased with increasing pCO 2 . The large range of metabolic rates observed at the single-cell level may reflect a response by colony-forming microbial populations to highly variable microenvironments.

Acidification of seawater caused by anthropogenic carbon dioxide (CO 2 ) is anticipated to influence the growth of dinitrogen (N 2 )-fixing phytoplankton, which contribute a large fraction of primary production in the tropical and subtropical ocean. We found that growth and N 2 -fixation of the ubiquitous cyanobacterium Trichodesmium decreased under acidified conditions, notwithstanding a beneficial effect of high CO 2 Acidification resulted in low cytosolic pH and reduced N 2 -fixation rates despite elevated nitrogenase concentrations. Low cytosolic pH required increased proton pumping across the thylakoid membrane and elevated adenosine triphosphate production. These requirements were not satisfied under field or experimental iron-limiting conditions, which greatly amplified the negative effect of acidification., (Copyright © 2017, American Association for the Advancement of Science.)