To protect the O2-sensitive nitrogenase enzyme, diazotrophic cyanobacteria separate N2 fixation from photosynthesis in space or time. However, in the filamentous Trichodesmium, technical limitations and conflicting results have so far prevented a consensus on the separation mechanism. Here, we specifically adapted single-cell methods for simultaneously capturing both photosynthesis and N2 fixation at the levels of gene expression, fixation and transport of carbon and nitrogen. Testing expression of nifH (nitrogenase) and psbA (photosystem II), we found that about half of the cells exclusively expressed either one of the genes at a given time, with nifH expression mostly in stretches of several consecutive cells within a filament. Short (30 min) stable isotope labelling enabled us to capture 25% of cells in a stage where they fixed nitrogen but not carbon. Lugol staining suggests that low O2 levels in these cells were maintained by glycogen respiration. Half of the cells received freshly fixed nitrogen within 30 min, most likely by rapid intercellular transport. Taken together, our findings suggest that single-cell regulation of gene expression in combination with rapid intercellular transport of freshly fixed nitrogen as well as glycogen respiration facilitate the separation of N2 fixation and photosynthesis in Trichodesmium.

Anthropogenic carbon dioxide emissions drive ocean acidification. Trichodesmium, a key marine nitrogen-fixing cyanobacterium, displays contrasting growth responses to ocean acidification across morphotypes: negative in filamentous free trichomes but neutral or positive in colonies. However, lacking mechanistic understanding for these discrepancies has impaired our ability to predict Trichodesmium's ecophysiological response. Here, we develop ecophysiological models to underpin mechanisms behind these divergent responses. For free trichomes, ocean acidification reduces nitrogen-fixing enzyme activity and photosynthetic energy production. In colonies, however, it alleviates copper and ammonia toxicity within the microenvironment-likely synergizing with enhanced iron acquisition-thereby outweighing minor benefit from relieved inorganic carbon limitation in the colony center. Projections suggest that globally, ocean acidification will reduce nitrogen fixation of trichomes by 16 +/- 6% but increase that of colonies by 19 +/- 24% within this century. By resolving morphotype-specific mechanisms, our study clarifies Trichodesmium's adaptive strategies for sustaining its competitiveness and biogeochemical impacts in the changing ocean.

Aerosol dust deposited on the nutrient-deprived surface ocean can boost phytoplankton growth and oceanic carbon uptake. Low mineral solubility restricts the biological utilization of dust-nutrients, thereby benefiting phytoplankton that actively dissolve dust. The ubiquitous colony-forming, N2-fixing cyanobacteria Trichodesmium specialize in dust-nutrient utilization, with several dust-dissolution pathways identified in natural populations. Studying active dust dissolution by Trichodesmium, we surveyed the elemental composition (i.e., quotas) of natural colonies from the dust-impacted Red Sea using a benchtop micro-x-ray fluorescence imager. We also examined changes in the colonies' quotas during incubations with dust and nutrients. Accounting for inter-colony variability, we analyzed 106 individual colonies. Since particles often appeared on the surface of colonies, we carefully analyzed all images, removing dust particle signals from colony quotas. Focusing on the colonies' iron (Fe) and phosphorus (P) quotas, we observed contrasting patterns of inter-colony variability and responses to dust, likely reflecting distinct nutrient sources-Fe sourced from dust and P sourced from the dissolved phase. Iron uptake from dust was repeatedly observed, but only upon colony-mineral interactions, indicative of contact-dependent active dissolution. The role of dust in Fe nutrition was also evident from the minor impact of dissolved Fe complexation on Fe quotas. Phosphorus quotas responded rapidly to P addition or removal, but not to dust. Natural colonies collected over a season had heterogeneous Fe quotas but homogeneous P quotas, further supporting their distinct sources. Predictions of Trichodesmium's bloom dynamics in particle-rich and dust-impacted ocean environments should incorporate its ability to dissolve Fe-minerals.

Here, we present a protocol for visualizing gene expression in the filamentous cyanobacterium Trichodesmium and the single-celled species Synechocystis and Cyanothece using the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique. We describe steps for fixation, agarose coating, enzymatic permeabilization, and sample handling. This protocol is broadly applicable to cyanobacteria, and the detection of rbcL mRNA in Trichodesmium, used as an example, supports its use to study the heterogeneity of physiological processes at single-cell level.

Trichodesmium, the predominant marine diazotrophic cyanobacterium, concurrently performs nitrogen (N2) fixation and photosynthesis, the latter of which produces oxygen (O2) that inhibits N2 fixation. Hopanoid lipids in Trichodesmium may play a role in dynamically regulating membrane permeability to O2, potentially alleviating O2 stress on N2 fixation. However, the physiological impacts of this dynamic permeability are not well understood. We developed a model showing that dynamically modulating membrane O2 permeability can enhance N2 fixation and growth of Trichodesmium by over 50%. High O2 permeability (1.5 × 10-4 of O2 diffusivity in seawater) during strong photosynthesis accelerates O2 exhaust, reducing energy-consuming photorespiration by ~40%, while low O2 permeability (1.0 × 10-5 diffusivity) during active N2 fixation minimizes O2 stress on N2 fixation. Together, these mechanisms increase the carbon and iron use efficiencies by ~70%. Our study provides a mechanistic and quantitative framework for how dynamic O2 permeability benefits Trichodesmium, offering insights potentially applicable to other diazotrophs.IMPORTANCETrichodesmium is a key player in marine N2 fixation, essential for oceanic productivity and global biogeochemical cycles. However, a significant challenge arises from the concurrent photosynthetic production of O2 during N2 fixation, which can inhibit N2 fixation and cause energy-wasting photorespiration. We develop a physiological model showing that Trichodesmium may dynamically regulate membrane O2 permeability to enhance N2 fixation and growth. The model suggests two mechanisms: elevated O2 permeability during the early daytime of strong photosynthesis accelerates O2 exhaust to the environment, reducing photorespiration, while reduced O2 permeability later limits O2 influx from the environment, lowering wasteful respiration and maintaining a low intracellular O2 level for active N2 fixation. These adaptations improve the efficiency of carbon and iron utilization, thereby facilitating N2 fixation and growth in Trichodesmium. This study sheds light on how Trichodesmium and other N2-fixing microorganisms can optimize their physiological processes in response to environmental challenges.

Colonies of the N-2-fixing cyanobacterium Trichodesmium spp. constitute a consortium with multiple microorganisms that collectively exert ecosystem-level influence on marine carbon and nitrogen cycling, shunting newly fixed nitrogen to low nitrogen systems, and exporting both carbon and nitrogen to the deep sea. Here we identify a seasonally recurrent association between puff colonies and amoebae through a two-year survey involving over 10 000 Trichodesmium colonies in the Red Sea. This association was most commonly found in near-shore populations during spring. Microscopic observations revealed consistent amoebae morphology throughout the study, and both morphological characteristics and 18S rRNA gene sequencing suggested that these amoebae are likely to belong to the species Trichosphaerium micrum, an amoeba that forms a CaCO3 shell. Co-cultures of Trichosphaerium micrum and Trichodesmium grown in the laboratory suggest that the amoebae feed on heterotrophic bacteria and not Trichodesmium, which adds a consumer dynamic to the complex microbial interactions within these colonies. Sinking experiments with fresh colonies indicated that the presence of the CaCO3-shelled amoebae decreased colony buoyancy. As such, this novel association may accelerate Trichodesmium sinking rates and facilitate carbon and nitrogen export to the deep ocean. Amoebae have previously been identified in Trichodesmium colonies in the western North Atlantic (Bermuda and Barbados), suggesting that this type of association may be widespread. This association may add a new critical facet to the microbial interactions underpinning carbon and nitrogen fixation and fate in the present and future ocean.

Photosynthesis which harvests energy from the Sun, whether it occurs in prokaryotes or eukaryotes, is a key biological process that ultimately nourishes the biosphere. The molecular efficiencies of the photo-physical and physiological processes are intricately tied not only to the photo-physics/enzymatic kinetics of the proteins involved, but also to their spatial co-localization in membrane microdomains or in cell compartments (e.g., in membrane-less organelles). Similar heterogeneity in function can be found also between cells in isogenic cell cultures (phenotypic heterogeneity) or in filaments of phototrophic cells (e.g., heterocysts/vegetative cells in nitrogen fixing cyanobacteria). This review paper delves into the connection between the spatial (co)-localization of biomolecules (lipids, RNA, DNA, proteins, membranes compartments) and their functionality in situ . We highlight recent methodological advances in the field (e.g., super-resolution microscopy, Raman micro-spectroscopy, nanoSIMS, microsensors) and showcase applications of these methods in understanding heterogeneity on single-cell and on population-scale level. This paper thus aims to highlight the avenues that will help to unravel the molecular, cellular and ecological mechanisms in photobiology by combining up-to-date microscopy techniques with more traditional functional approaches.

The marine cyanobacterium Trichodesmium has a remarkable ability to interact with and utilize air-borne dust as a nutrient source. However, dust may adversely affect Trichodesmium through buoyancy loss and exposure to toxic metals. Our study explored the effect of desert dust on buoyancy and mortality of natural Red Sea puff-shaped Trichodesmium thiebautii . Sinking velocities and ability of individual colonies to stay afloat with increasing dust loads were studied in sedimentation chambers. Low dust loads of up to ~400 ng per colony did not impact initial sinking velocity and colonies remained afloat in the chamber. Above this threshold, sinking velocity increased linearly with the colony dust load at a slope matching prediction based on Stoke’s law. The potential toxicity of dust was assessed with regards to metal dissolution kinetics, differentiating between rapidly released metals that may impact surface blooms and gradually released metals that may impact dust-centering colonies. Incubations with increasing dust concentrations revealed colony demise, but the observed lethal dose far exceeded dust concentrations measured in coastal and open ocean systems. Removal of toxic particles as a mechanism to reduce toxicity was explored using SEM-EDX imaging of colonies incubated with Cu-minerals, yet observations did not support this pathway. Combining our current and former experiments, we suggest that in natural settings the nutritional benefits gained by Trichodesmium via dust collection outweigh the risks of buoyancy loss and toxicity. Our data and concepts feed into the growing recognition of the significance of dust for Trichodesmium ’s ecology and subsequently to ocean productivity.

The N2-fixing cyanobacterium Trichodesmium is an important player in the oceanic nitrogen and carbon cycles. Trichodesmium occurs both as single trichomes and as colonies containing hundreds of trichomes. In this review, we explore the bene-fits and disadvantages of colony formation, considering physical, chemical, and biological effects from nanometer to kilometer scale. Showing that all major life challenges are affected by colony formation, we claim that Trichodesmium's eco-logical success is tightly linked to its colonial lifestyle. Microbial interactions in the microbiome, chemical gradients within the colony, interactions with particles, and elevated mobility in the water column shape a highly dynamic microenvironment. We postulate that these dynamics are key to the resilience of Trichodesmium and other colony formers in our changing environment.

The Trichodesmium genus comprises some of the most abundant N2-fixing organisms in oligotrophic marine ecosystems. Since nitrogenase, the key enzyme for N2 fixation, is irreversibly inhibited upon O2 exposure, these organisms have to coordinate their N2-fixing ability with simultaneous photosynthetic O2 production. Although being the principal object of many laboratory and field studies, the overall process of how Trichodesmium reconciles these two mutually exclusive processes remains unresolved. This is in part due to contradictory results that fuel the Trichodesmium enigma. In this review, we sift through methodological details that could potentially explain the discrepancy between findings related to Trichodesmium's physiology. In doing so, we exhaustively contrast studies concerning both spatial and temporal nitrogenase protective strategies, with particular attention to more recent insights. Finally, we suggest new experimental approaches for solving the complex orchestration of N2 fixation and photosynthesis in Trichodesmium.

Low iron (Fe) and phosphorus (P) ocean regions are often home to the globally important N 2 ‐fixing cyanobacterium Trichodesmium spp., which are physiologically adapted to Fe/P co‐limitation. Given Trichodesmium 's eminent ability to capture particles and the common associations between Fe and P in sediments and aerosols, we hypothesized that mineral bio‐dissolution by Trichodesmium spp. may enable them to co‐acquire Fe and P. We present a new sensitive assay to determine P uptake from particles, utilizing 33 P‐labeled ferrihydrite. To validate the method, we examined single natural Trichodesmium thiebautii colonies in a high‐resolution radiotracer ß‐imager, identifying strong colony‐mineral interactions, efficient removal of external 33 P‐labeled ferrihydrite, and elevated 33 P uptake in the colony core. Next, we determined bulk P uptake rates, comparing natural Red Sea colonies and P‐limited Trichodesmium erythraeum cultures. Uptake rates by natural and cultured Trichodesmium were similar to P release rates from the mineral, suggesting tight coupling between dissolution and uptake. Finally, synthesizing P‐ferrihydrite labeled with either 33 P or 55 Fe, we probed for Fe/P co‐extraction by common microbial mineral solubilization pathways. Dissolution rates of ferrihydrite were accelerated by exogenous superoxide and strong Fe‐chelator and subsequently enhanced 33 P release and uptake by Trichodesmium . Our method and findings can facilitate further Fe/P co‐acquisition studies and highlight the importance of biological mechanisms and microenvironments in controlling bioavailability and nutrient fluxes from particles.

The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment.

Biological dinitrogen (N2) fixation is performed solely by specialized bacteria and archaea termed diazotrophs, introducing new reactive nitrogen into aquatic environments. Conventionally, phototrophic cyanobacteria are considered the major diazotrophs in aquatic environments. However, accumulating evidence indicates that diverse non-cyanobacterial diazotrophs (NCDs) inhabit a wide range of aquatic ecosystems, including temperate and polar latitudes, coastal environments and the deep ocean. NCDs are thus suspected to impact global nitrogen cycling decisively, yet their ecological and quantitative importance remain unknown. Here we review recent molecular and biogeochemical evidence demonstrating that pelagic NCDs inhabit and thrive especially on aggregates in diverse aquatic ecosystems. Aggregates are characterized by reduced-oxygen microzones, high C:N ratio (above Redfield) and high availability of labile carbon as compared to the ambient water. We argue that planktonic aggregates are important loci for energetically-expensive N2 fixation by NCDs and propose a conceptual framework for aggregate-associated N2 fixation. Future studies on aggregate-associated diazotrophy, using novel methodological approaches, are encouraged to address the ecological relevance of NCDs for nitrogen cycling in aquatic environments.

Photosynthesis and respiration cause distinct chemical microenvironments within cyanobacterial aggregates. Here, we used microsensors and a diffusion–reaction model to characterize gradients in carbonate chemistry and investigate how these are affected by ocean acidification in Baltic vs. Pacific aggregates (Nodularia and Dolichospermum vs. Trichodesmium). Microsensor measurements of O2 and pH were performed under in situ and expected future pCO2 levels on Nodularia and Dolichospermum aggregates collected in the Baltic Sea. Under in situ conditions, O2 and pH levels within the aggregates covered ranges of 80–175% air saturation and 7.7–9.4 in dark and light, respectively. Carbon uptake in the light was predicted to reduce HCO3− by 100–150 μmol L−1 and CO2 by 3–6 μmol L−1 in the aggregate center compared to outside, inducing strong CO2 depletion (down to 0.5 μmol L−1 CO2 remaining in the center) even when assuming that HCO3− covered 80–90% of carbon uptake. Under ocean acidification conditions, enhanced CO2 availability allowed for significantly lower activity of carbon concentrating mechanisms, including a reduction of the contribution of HCO3− to carbon uptake by up to a factor of 10. The magnification of proton gradients under elevated pCO2 that was predicted based on a lower buffer capacity was observed in measurements despite a concurrent decrease in photosynthetic activity. In summary, we provide a quantitative image of the inorganic carbon environment in cyanobacterial aggregates under present‐day and expected future conditions, considering both the individual and combined effects of the chemical and biological processes that shape these environments.

[This corrects the article DOI: 10.1016/j.csbj.2021.11.036.].

Stable isotope probing (SIP) combined with nano-scale secondary ion mass spectrometry (nanoSIMS) is a powerful approach to quantify assimilation rates of elements such as C and N into individual microbial cells. Here, we use mathematical modeling to investigate how the derived rate estimates depend on the model used to describe substrate assimilation by a cell during a SIP incubation. We show that the most commonly used model, which is based on the simplifying assumptions of linearly increasing biomass of individual cells over time and no cell division, can yield underestimated assimilation rates when compared to rates derived from a model that accounts for cell division. This difference occurs because the isotopic labeling of a dividing cell increases more rapidly over time compared to a non-dividing cell and becomes more pronounced as the labeling increases above a threshold value that depends on the cell cycle stage of the measured cell. Based on the modeling results, we present formulae for estimating assimilation rates in cells and discuss their underlying assumptions, conditions of applicability, and implications for the interpretation of intercellular variability in assimilation rates derived from nanoSIMS data, including the impacts of storage inclusion metabolism. We offer the formulae as a Matlab script to facilitate rapid data evaluation by nanoSIMS users.

Temperature is one of the environmental factors that most strongly influence the life of microalgae, from the enzymatic level up to the modification of biosynthetic and degradation potential and, thus, cellular composition. When exposed to a change in environmental milieu two main responses can be attained by cells: acclimation or homoeostasis. Recent studies testing the impact of other environmental factors indicated that the choice between these strategies is related to growth rates, i.e. cells dividing at a faster rate showed inclination to acclimation while cells dividing at a slower rate were more prone to homoeostasis. These findings motivated us to further explore the extent to which these compositional response modes are applied under varying temperature. Two representative marine diatoms, one centric, Thalassiosira pseudonana, and one pennate, Cylindrotheca fusiformis were investigated. Species were cultured at 15 degrees C, 20 degrees C and 30 degrees C and at two growth rates. Compositional response was analysed with respect to elemental stoichiometry (carbon, nitrogen, silica, phosphorus, sulphur, manganese, iron, zinc and copper) and the major organic pool ratios (carbohydrate:lipid, carbohydrate:protein and protein:lipid). While T. pseudonana exhibited an acclimation response to temperature, C. fusiformis proved to be strongly homoeostatic. In the case of T. pseudonana, our data showed that the variations in composition were consistent with the growth rate dependence hypothesis for many micronutrients, but not for carbon and nitrogen contents, and C:N ratio. These species-specific differences in response mode highlight the diversity in ecophysiological strategies of diatoms, which needs to be considered when predicting climate change responses.

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.