The physiological cost of diazotrophy for Trichodesmium erythraeum IMS101

Abstract

Trichodesmium plays a significant role in the oligotrophic oceans, fixing nitrogen in an area corresponding to half of the Earth’s surface, representing up to 50% of new production in some oligotrophic tropical and subtropical oceans. Whilst Trichodesmium blooms at the surface exhibit a strong dependence on diazotrophy, colonies at depth or at the surface after a mixing event could be utilising additional N-sources. We conducted experiments to establish how acclimation to varying N-sources affects the growth, elemental composition, light absorption coefficient, N₂ fixation, PSII electron transport rate and the relationship between net and gross photosynthetic O₂ exchange in T. erythraeum IMS101. To do this, cultures were acclimated to growth medium containing NH₄⁺ and NO₃^- (replete concentrations) or N₂ only (diazotrophic control). The light dependencies of O₂ evolution and O₂ uptake were measured using membrane inlet mass spectrometry (MIMS), while PSII electron transport rates were measured from fluorescence light curves (FLCs). We found that at a saturating light intensity, Trichodesmium growth was ~ 10% and 13% lower when grown on N₂ than with NH₄⁺ and NO₃^-, respectively. Oxygen uptake increased linearly with net photosynthesis across all light intensities ranging from darkness to 1100 μmol photons m^-2 s^-1. The maximum rates and initial slopes of light response curves for C-specific gross and net photosynthesis and the slope of the relationship between gross and net photosynthesis increased significantly under non-diazotrophic conditions. We attribute these observations to a reduced expenditure of reductant and ATP for nitrogenase activity under non-diazotrophic conditions which allows NADPH and ATP to be re-directed to CO₂ fixation and/or biosynthesis. The energy and reductant conserved through utilising additional N-sources could enhance Trichodesmium’s productivity and growth and have major implications for its role in ocean C and N cycles.

Introduction

In marine ecosystems, phytoplankton primary production is often limited by the bioavailability of fixed N [1–3], where N-sources (e.g. NO₃^-, NO₂^-, NH₄⁺, urea etc) are quickly depleted by fast growing phytoplankton [4]. A significant fraction (~ 25 Tg N yr^-1) of N in the euphotic zone is lost via sedimentation to the deep ocean as particulate organic nitrogen (PON), making NO₃^- concentrations higher at greater depth [5–7]. Whilst areas of upwelling transport NO₃^- into the euphotic zone, there are vast regions of the oligotrophic open oceans that are dependent on the input of new N from N₂-fixing cyanobacteria. Among the most important marine diazotrophs are Trichodesmium sp., which can form extensive surface blooms in the tropical and subtropical oceans [8–12].

Previous studies have highlighted Trichodesmium’s capacity to assimilate various forms of combined N-sources [13–17]. It is commonly assumed that Trichodesmium obtains most of its nitrogen quota from N₂ fixation, however field-based measurements of N₂ fixation show wide temporal and spatial variability [18]. The causes of this variability remain unclear, but environmental factors such as the availability of combined nitrogen may be a contributing factor.

Diazotrophy

Diazotrophic cyanobacteria are able to meet their daily nitrogen quota by fixing dinitrogen (N₂).

$${\mathrm{N}}_2+16\mathrm{A}\mathrm{T}\mathrm{P}+{8\mathrm{H}}^{+}+{8\mathrm{e}}^{-}\to {2\mathrm{N}\mathrm{H}}_3+{\mathrm{H}}_2+16\mathrm{A}\mathrm{D}\mathrm{P}+16\mathrm{P}\mathrm{i}$$ (1)

While N₂ fixation is an extremely energy demanding process, Trichodesmium incurs additional costs related to the protection of nitrogenase from the irreversible inhibition of photosynthetically evolved O₂ [9, 19, 20]. The separation of O₂ evolution and N₂ fixation is regulated over a diurnal cycle of N₂ fixation and photosynthesis [21], involving daily synthesis and degradation of nitrogenase [22, 23] and alternation of photosynthetic activity states [24]. Temporal separation occurs over short timescales, where peak rates of photosynthesis (~ 10 am) and N₂ (~ 12 pm) fixation vary over a diel period. Spatial separation occurs via diazocytes, which are reversibly specialised cells for nitrogen fixation [25, 26]. Diazocytes contain the necessary proteins to perform photosynthetic CO₂ fixation and N₂ fixation. However, it has been suggested that when fixing N₂, cells increase cyclic electron transport around PSI to enhance ATP synthesis [21, 24], thus allowing the cells to meet the energetic demands of N₂ fixation (Eq 1).

Uptake of additional N-sources

Like other facultative diazotrophic cyanobacteria spp., Trichodesmium can exploit other forms of nitrogen including NH₄⁺, NO₃^-, urea and amino acids [16, 27]. These N compounds are transported into the cell via permeases, metabolised to NH₄⁺ and then incorporated into carbon skeletons through the glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT) pathways. This process is mediated by nitrate reductase (Eq 2) and nitrite reductase (Eq 3).

$${\mathrm{N}\mathrm{O}}_3^{-}+{2\mathrm{e}}^{-}+{2\mathrm{H}}^{+}\to {\mathrm{N}\mathrm{O}}_2^{-}+{\mathrm{H}}_2\mathrm{O}$$ (2)

$${\mathrm{N}\mathrm{O}}_2^{-}+{6\mathrm{e}}^{-}+{8\mathrm{H}}^{+}\to {\mathrm{N}\mathrm{H}}_4^{+}+{2\mathrm{H}}_2\mathrm{O}$$ (3)

For cyanobacteria, nitrate reductase is located in the cytosol and uses NADPH to catalyse the transfer of two electrons. The NO₂^- formed by nitrate reductase is further reduced to NH₄⁺ via the transfer of six electrons. Thus, the reduction of NO₃^- to NH₄⁺ can be expressed as;

$${\mathrm{N}\mathrm{O}}_3^{-}+{8\mathrm{e}}^{-}+{10\mathrm{H}}^{+}\to {\mathrm{N}\mathrm{H}}_4^{+}+{3\mathrm{H}}_2\mathrm{O}$$ (4)

Amino acids are synthesised from ammonia (NH₃) via the GS-GOGAT pathway. The initial GS pathway requires ATP and glutamate as a substrate;

$$\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{a}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{e}+{\mathrm{N}\mathrm{H}}_3+\mathrm{A}\mathrm{T}\mathrm{P}\to \mathrm{G}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+\mathrm{A}\mathrm{D}\mathrm{P}+\mathrm{P}\mathrm{i}$$ (5)

where glutamine is subsequently transformed to 2-oxoglutarate and reduced using NADPH, forming two moles of glutamate.

$$2-\mathrm{O}\mathrm{x}\mathrm{o}\mathrm{g}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}+\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\to 2\left[\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{a}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{e}\right]$$ (6)

Thus, for every mole of glutamate produced, one mole each of NH₃, NADPH, ATP and 2-oxoglutarate are required. Additionally, ATP is required for the active transport of inorganic NH₄⁺ or NO₃^- into the cell [28]. Different N-sources require different amounts of energy and reductant and as such can be ordered into a hierarchy of energy requirements; where diazotrophy requires the highest investment of electrons and ATP, followed by NO₃^-, NO₂^- and then NH₃.

Utilising additional N-sources

Global warming is increasing sea surface temperatures (SSTs) which is enhancing water stratification and decreasing vertical mixing [29], potentially increasing the area of N-limited oceans. Whilst detrimental to many phytoplankton, a reduced flux of NO₃^- into the upper mixed layer will increase the competitive advantage of diazotrophs for other limiting nutrients (i.e. Fe or P). Trichodesmium colonies have been observed migrating to the nutricline [30, 31] to facilitate the luxury uptake of polyphosphates before returning to the surface. Whilst at these depths, cells are exposed to NO₃^- concentrations greater than those at the surface. As such, Trichodesmium colonies may be assimilating and storing (i.e. cyanophycin granules) more combined N than the blooms frequently measured on the surface [32]. This could have major implications for growth rates, primary productivity and biogeochemical cycles [33].

Our approach comprises a systematic experiment where T. erythraeum IMS101 was grown over long durations, at three N-source treatments, with controlled and well-defined growth conditions, ensuring fully acclimated, balanced growth had been achieved. Our aims were to assess the response of T. erythraeum IMS101 growth, light dependency of gross and net O₂ photosynthesis, PSII electron transport rates and elemental composition to different N-sources; investigating the physiological cost of performing diazotrophy.

Materials and methods

T. erythraeum IMS101 was semi-continuously cultured to achieve fully acclimated balanced growth at three N-source treatments (N₂, NH₄⁺ and NO₃^-), at a targeted 380 μatm CO₂ concentration, saturating light intensity (400 μmol photons m^-2 s^-1), 12:12 light:dark (L:D) cycle and optimal temperature (26 °C ± 0.2) (3 treatments in total) for ~ 2 months (~ 30 generations).

Experimental setup

Cultures were acclimated to the CO₂ and light intensity for ~ 4 months (~ 60 generations) under diazotrophic conditions before the addition of NH₄⁺ or NO₃^-. Cultures were gradually enriched over a 2/3-week period by increasing the dilution ratio of YBCII media containing NH₄⁺ or NO₃^- (100 μM).

T. erythraeum IMS101 was grown using YBCII medium [34] at 1.5 L volumes in 2 L pyrex bottles that were acid-washed and autoclaved prior to culturing. Daily growth rates were quantified from changes in baseline fluorescence (F_o) measured between 09:00 to 10:30 on dark-adapted cultures (20 minutes) using a FRRfII FastAct Fluorometer System (Chelsea Technologies Group Ltd, UK). Cultures were regarded as fully acclimated and in balanced growth when both the slope of the linear regression of ln F_o versus time and the ratio of live cell to acetone extracted (method detailed below) baseline fluorescence (F_o) were constant following every dilution with fresh YBCII medium. Cultures were kept at the upper section of the exponential growth phase through periodic dilution with new growth media at 3–5 day intervals. Illumination was provided side-on by fluorescent tubes (Sylvania Luxline Plus FHQ49/T5/840). Cultures were constantly mixed using magnetic PTFE stirrer bars and aerated with a filtered (0.2μm pore) air mixture at a rate of ~ 200 mL s^-1. The CO₂ concentration was regulated (± 2 μatm) by mass flow controllers (Bronkhorst, Newmarket, UK). CO₂-free air was supplied by an oil free compressor (Bambi Air, UK) via a soda lime gas-tight column which was mixed with a 10% CO₂ in-air mixture from a gas cylinder (BOC Industrial Gases, UK). The CO₂ concentration was continuously monitored and recorded by an infra-red gas analyser (Li-Cor Li-820, Nebraska USA), calibrated weekly by a standard gas (BOC Industrial Gases).

Throughout all culturing, the inorganic carbon chemistry (S1 File) and dissolved inorganic NH₄⁺ and NO₃^- concentrations (S2 File) were determined prior to diluting with fresh media. Samples for elemental composition, photosynthesis-light response curves, fluorescence light curves (FLC), in vivo light absorption and acetylene reduction assays were collected at the same time of day, approximately 4 and 6 hours into the photo-phase of the L:D cycle.

Measuring O₂ exchange by membrane inlet mass spectrometry (MIMS)

Light dependent rates of O₂ production and consumption were measured with a membrane inlet mass spectrometer (MIMS), using an ¹⁸O₂ technique modified from McKew et al. [35] (S3 File). MIMS measurements consisted of three biological replicates per treatment (S4 File). Chlorophyll a concentrations at the point of sampling ranged from 80 to 245 μg Chla L^-1.

Changes in ¹⁶O₂ and ¹⁸O₂ and thus O₂ consumption (U_o) and O₂ evolution (E_o) were calculated using the following equations [36];

$$U_0=-\left(1+\frac{{{}_{}{}^{16}O}_2}{{{}_{}{}^{18}O}_2}\right)\bullet \frac{\Delta {{}_{}{}^{18}O}_2}{\Delta t}$$ (7)

$$E_0=\frac{\Delta {{}_{}{}^{16}O}_2}{\Delta t}-\left(\frac{{{}_{}{}^{16}O}_2}{{{}_{}{}^{18}O}_2}\right)\bullet \frac{\Delta {{}_{}{}^{18}O}_2}{\Delta t}$$ (8)

where U_o is the rate of O₂ consumption calculated from the decrease of ¹⁸O₂ over time (i.e. Δ ¹⁸O₂/Δt), which takes into account the relative concentration of ¹⁸O₂ compared to ¹⁶O₂ (i.e. 1 + ¹⁶O₂/¹⁸O₂) and E_o is the rate of gross O₂ evolution calculated from the increase in ¹⁶O₂ over time (Δ¹⁶O₂/Δt), where the decline of ¹⁸O₂ (i.e. Δ¹⁸O₂/Δt) and ¹⁸O₂ is corrected for relative to the concentration of ¹⁶O₂. Chlorophyll a- and C-specific rates were obtained by dividing U₀ and E₀ by the concentration of Chla and particulate organic carbon, respectively. Rates were multiplied by 1.073 to spectrally correct to the culturing LEDs (S1 Fig).

Photosynthesis-light (P-E) curves for gross (E₀^Chl(C)) and net photosynthesis (P_net^Chl(C) = E₀^Chl(C)-U₀^Chl(C)) were fitted to the equations from Platt and Jassby [37];

$$E_0^{Chl\left(C\right)}=E_{0m}^{Chl\left(C\right)}·\left[1-e\left(\frac{-{\alpha }_g^{Chl\left(C\right)}\bullet E}{E_{0m}^{Chl\left(C\right)}}\right)\right]$$ (9)

$${P_{net}}_{}^{Chl\left(C\right)}={P_{net}}_m^{Chl\left(C\right)}·\left[1-e\left(\frac{-{\alpha }_n^{Chl\left(C\right)}\bullet E}{{P_{net}}_m^{Chl\left(C\right)}}\right)\right]+R_d^{Chl\left(C\right)}$$ (10)

where E_0m^Chl(C) and P_netm^Chl(C) are the maximum gross and net O₂ evolution rates; α_g ^Chl(C) and α_n^Chl(C) are the initial light-limited slopes for gross and net photosynthesis; R_d is the dark respiration rate; and E is the light intensity (μmol photons m^-2 s^-1). Curve fitting was performed on each replicate separately to calculate mean (± S.E.) curve fit parameterisations (Sigmaplot 11.0).

The maximum quantum efficiency of gross (ɸ_mgross) and net (ɸ_mnet) O₂ evolution was calculated as follows;

$${\mathrm{ɸ}}_m=\frac{{\alpha }_{g\left(n\right)}^C}{a_{eff}^C}$$ (11)

where the C-specific initial slope for gross (α_g^C) or net (α_n^C) O₂ evolution was divided by the C-specific, effective light absorption coefficient (a_eff^C).

Measuring nitrogenase activity by acetylene reduction

Acetylene reduction rates were measured using gas chromatography (ATI Unicam 610 series). Gaseous samples were injected into the GC column head (60 °C), carried via N₂ gas through a Porapak N column (100 °C) to a flame ionising detector (100 °C). Peak areas of acetylene and ethylene were quantified by an integrated chromatograph data acquisition unit (Shimadzu C-R8A Integrator) and were converted into concentrations via an acetylene and ethylene standard curve performed with standard gases (Scientific and Technical Gases Ltd., UK). Triplicate 6 mL samples of each biological replicate culture were placed into 12 mL exetainer, screw capped glass vials (Labco Ltd, UK). Exactly 1.2 mL of the headspace was removed and replaced with a 1.2 mL sample of acetylene (BOC Industrial Gases, UK) (headspace = 20% acetylene). The vials were gently inverted for 1 minute before 250 μL of headspace was injected into the GC column for an initial measurement of acetylene and ethylene concentrations (T₀). Vials were incubated at 26 °C and 400 μmol photons m^-2 s^-1 in an aluminium temperature block and were gently inverted every 10 minutes to prevent trichomes from settling on the bottom or aggregating at the meniscus. After 1 hour, a second 250 μL gaseous headspace was injected into the GC column for the post-incubation measurement (T₁). Temperature and pressure was measured during each set of measurements and accounted for in the calculations. The rate of ethylene production was calculated with the assumption that the concentrations of acetylene and ethylene within the media were always in equilibrium to those in the headspace;

$${\Delta C}_2{H}_2=\frac{{C_2{H}_2}_{\left(T_1\right)}-{C_2{H}_2}_{\left(T_0\right)}}{t\bullet V_{\left(I\right)}}$$ (12)

where (ΔC₂H₂) is the ethylene production rate (μmol C₂H₄ h^-1), C₂H_2(T0) and C₂H_{2 (T1)} are the ethylene concentrations in the headspace at the start (T₀) and end (T₁) of the incubation, V_(I) is the volume of gaseous sample injected into the GC column (L^-1) and t is the incubation time (min).

N₂ fixation rates were calculated to a Chla (μmol N₂ (mg Chla)^-1 h^-1) and total carbon (μmol N₂ (mg C)^-1 h^-1) basis;

$$N_{2}fixation=\left(\frac{{\Delta C}_2{H}_2}{\left[Chla\left(C\right)\right]}\bullet {10}^3\right)\bullet 0.25$$ (13)

where ΔC₂H₂ (μmol h^-1) is divided by the Chla or total carbon concentration (mg) and multiplied by 0.25 under the assumption that reduction of four moles of acetylene is equivalent to reduction of one mole of dinitrogen.

Fluorescence light curves (FLCs)

A 2 mL sample of each replicate culture was used to measure a fluorescence light curve (FLC) [38]. The FLCs were measured with a FRRfII FastAct Fluorometer System, using a white LED actinic light source (Chelsea Technologies Group Ltd, UK). Each FLC lasted 1 hour; comprising 12 light steps which ranged from 10 to 1600 μmol photon m^-2 s^-1, each lasting 5 minutes in duration. The FLCs provided measurements of the light absorption cross-section of PSII photochemistry (σ_PIIʹ), the average time constant for the re-opening of a closed PSII reaction centre (τ_fʹ) and the operating efficiency of PSII photochemistry (F_qʹ/F_mʹ);

$$\frac{F_q\mathrm{ʹ}}{F_m\mathrm{ʹ}}=\left[\frac{F_m\mathrm{ʹ}-F\mathrm{ʹ}}{F_m\mathrm{ʹ}}\right]$$ (14)

where F_mʹ is the maximum fluorescence in the light-adapted state and Fʹ is the steady-state fluorescence at any point.

Photosystem II (PSII) electron transport rates were normalised to a Chla (mol e^- (g Chla)^-1 h^-1) and total carbon (mol e^- (g C)^-1 h^-1) basis;

$${ETR}^{Chl\left(C\right)}=\frac{F_q\mathrm{ʹ}}{F_m\mathrm{ʹ}}\bullet E\bullet \left(a^{Chl\left(C\right)}·{FAQ}_{PII}\right)\bullet 3600\bullet SCF$$ (15)

where F_qʹ/F_mʹ is the operating efficiency of PSII photochemistry; E is the light intensity (mol photons m^-2 s^-1), a^Chl(C) is the Chla-specific (C-specific) effective light absorption (m² g^-1 Chla and m² g^-1 C, respectively), FAQ_PII is the fraction of absorbed photons directed to PSII, which was set to 0.5 [39], with the assumption that the quantum yield of electron transport of one trapped photon within a reaction centre is equal to 1 [40]; 3600 converts seconds to hours and SCF is a spectral correction factor of 1.194, which converts electron transport rates to the culturing LED spectrum (S1 Fig).

ETR curves were modelled using a P-E equation [37], performed on each individual replicate using a Marquardt–Levenberg least squares algorithm to generate the best fit (R² > 0.993);

$$\mathrm{E}\mathrm{T}\mathrm{R}={\mathrm{E}\mathrm{T}\mathrm{R}}_m\mathrm{ʹ}\bullet \left[1-e\left(\frac{-{\alpha }_{ETR}\bullet E}{{\mathrm{E}\mathrm{T}\mathrm{R}}_m\mathrm{ʹ}}\right)e\left(\frac{-{\beta }_{ETR}\bullet E}{{\mathrm{E}\mathrm{T}\mathrm{R}}_m\mathrm{ʹ}}\right)\right]$$ (16)

where ETR_mʹ is the hypothetical Chla(C)-specific maximum electron transport rate that would be achieved if there was no photoinhibition (mol e^- (g Chla(C))^-1 h^-1); α_ETR is the initial slope of the Chla(C)-specific ETR-light curve (mol e^- (g Chla(C))^-1 h^-1 (μmol photons m^-2 s^-1)^-1); β_ETR is the parameter that accounts for downregulation and/or photoinhibition at supra-optimal light intensities (mol e^- (g Chla(C))^-1 h^-1 (μmol photons m^-2 s^-1)^-1); and E is the light intensity (μmol photons m^-2 s^-1).

The realised maximum PSII electron transport rate in the presence of photoinhibition (ETR_m), light intensity at which ETR is maximal (E_opt), the light-saturation parameter (E_k) and the light inhibition parameter (E_p) were calculated from the fitted parameters as follows:

$${ETR}_m={ETR}_m\mathrm{\text{'}}\bullet \left(\frac{{\alpha }_{ETR}}{{\alpha }_{ETR}+{\beta }_{ETR}}\right)\bullet {\left(\frac{{\beta }_{ETR}}{{\alpha }_{ETR}+{\beta }_{ETR}}\right)}^{\frac{{\beta }_{ETR}}{{\alpha }_{ETR}}}$$ (17)

$$E_{opt}=\frac{{ETR}_m\mathrm{\text{'}}}{{\alpha }_{ETR}}\bullet ln\left(\frac{{\alpha }_{ETR}+{\beta }_{ETR}}{{\beta }_{ETR}}\right)$$ (18)

$$E_k=\frac{{ETR}_m}{{\alpha }_{ETR}}$$ (19)

$$E_p=\frac{{ETR}_m}{{\beta }_{ETR}}$$ (20)

The ratio of PSII electron transport to gross O₂ evolution (E₀) under light-limitation (Φ_eα) and light-saturation (Φ_em) were calculated as follow;

$${\mathrm{\Phi }}_{\mathrm{e}\mathrm{\alpha }}=\frac{{\alpha }_{ETR}}{{\alpha }_g}$$ (21)

$${\mathrm{\Phi }}_{\mathrm{e}\mathrm{m}}=\frac{{ETR}_m}{E_{0m}}$$ (22)

Cellular elemental composition and light absorption

Samples for determining particulate organic carbon (POC), nitrogen (PN) and phosphorus (PP) (S5 File), chlorophyll a (S6 File) and in vivo light absorption (S7 File) were collected with each MIMS measurement, with each sample being a biological replicate.

Modelling the in vivo light absorption from pigment absorption spectra

In vivo light absorption was reconstructed using the light absorption spectra of Chla and photoprotective carotenoids (PPC) taken from Woźniak et al. [41] and the light absorption spectra of phycourobilin (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2) taken from Küpper et al. [42] (S2 Fig).

The Chla-specific light absorption coefficient was modelled as the sum of the contribution of all pigments;

$$a_{mod}^{Chl}\left(\lambda \right)={\sum }_i\bullet {\beta }_i\bullet a_i\left(\lambda \right)$$ (23)

where a^Chl_mod is the modelled in vivo light absorption at a specific wavelength (λ = 400–700 nm); βⁱ is the contribution of each pigment to a^Chl_mod and aⁱ is the pigment-specific spectral absorption coefficient of pigment i, in m² (g pigment i)^-1.

The modelled in vivo light absorption spectra (a^Chl_mod (λ)) was optimised to the measured spectra between 400 and 700 nm using a reduced sum of squares method (Sigmaplot 11.0). If a zero value was returned for a βⁱ parameter, that pigment was removed from the model and the curve fit reapplied.

Results

Inorganic C-chemistry, growth rate and cell composition

Balanced growth of T. erythraeum IMS101 was 0.34 d^-1 when grown on N₂, increasing by 10% and 13% when grown in the presence of NH₄⁺ and NO₃^-, respectively (Table 1). Particulate C:N, C:P and N:P ratios were all influenced by the presence of additional N-sources. When compared to the N₂ treatment, C:N decreased by 36% and 43% for the NH₄⁺ and NO₃^- treatments, respectively. Ratios of C:P and N:P were comparable between NH₄⁺ and NO₃^- treatments, but were significantly lower (~ 60% and 35%, respectively) compared to the N₂ treatment (Table 1). Ratios of Chla:C were 80% and 67% higher for the NH₄⁺ and NO₃^- treatments than for the N₂ treatment, while Chla:N was not significantly different between treatments (Table 1). Carbon and Chla-specific N₂ fixation rates were highest for the N₂ treatment, decreasing significantly by 84% and 80% (Chla-specific) and 73% and 68% (C-specific) for the NH₄⁺ and NO₃^- treatments, respectively (Table 1).

Table 1. The median (± S.E.) balanced growth rates and mean elemental stoichiometry and N₂ fixation rates for T. erythraeum IMS101 when acclimated to three N-source conditions (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

Variables	Units	N2	NH4+	NO3-
Growth rate	d-1	0.340 (0.038)[A]	0.375 (0.011)[B]	0.384 (0.005)[B]
Elemental Stoichiometry
C:N	mol:mol	6.9 (0.7)	4.4 (0.9)	3.9 (0.7)
C:P	mol:mol	122.6 (7.0)[B]	47.9 (2.4)[A]	36.9 (2.9)[A]
N:P	mol:mol	18.1 (1.3)[B]	11.8 (2.2)	9.9 (1.0)[A]
Chla:C	mg:mol	134 (8)[A]	239 (4)[B]	222 (2)[B]
Chla:N	mg:mol	906 (43)	1041 (209)	855 (154)
N2 Fixation
Chla-specific	μmol N (mg Chla)-1 h-1	14.75 (1.66)[B]	2.35 (0.49)[A]	2.84 (0.44)[A]
C-specific	μmol N (mg C)-1 h-1	0.16 (0.01)[B]	0.04 (0.01)[A]	0.05 (0.01)[A]

Abbreviations; C:N, C:P and N:P ratios are mol:mol, Chla:C and Chla:N ratios are mg:mol (n = 3). Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A].

The inorganic carbon concentration, pH and alkalinity (A_T) did not vary significantly amongst N-source treatments. Overall, CO₂ drawdown ranged between 78 to 92 μatm from the target concentration (i.e. 380 μatm) for all N-source treatments (Table 2) and exhibited little variability over a diurnal cycle (S3 Fig). Inorganic N concentrations were > 1 μM for the N₂ treatment and were ~ 8 μM for the NH₄⁺ and NO₃^- treatments at the point of dilution (Table 2).

Table 2. The growth conditions (± S.E.) for T. erythraeum IMS101 when cultured under three N-source conditions (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

Variables	Units	N2	NH4+	NO3-
pH	Total	8.18	8.18	8.19
H+	nM	6.6 (0.1)	6.6 (0.1)	6.4 (0.2)
AT	μM	2483 (47)	2427 (59)	2482 (56)
TCO2	μM	2066 (41)	2019 (51)	2056 (44)
HCO3-	μM	1762 (35)	1723 (44)	1746 (33)
CO32-	μM	296 (7)	288 (9)	302 (12)
CO2	μM	8.3 (0.2)	8.2 (0.3)	8.0 (0.4)
pCO2	μatm	300 (8)	296 (9)	289 (7)
NH4+	μM	0.76 (0.13)	8.33 (0.45)	0.59 (0.07)
NO3-	μM	0.07 (0.07)	0.46 (0.07)	8.24 (1.31)
n		35	34	10

Individual pH values were converted to a H⁺ concentration, allowing a mean pH value to be calculated.

Light absorption

The effective light absorption coefficients were not significantly different between N-source treatments, nor were the modelled absorption coefficients significantly different to the measured coefficients; with modelled coefficients being only 1 to 3% higher across all N-source treatments (Table 3).

Table 3. The mean (± S.E.) measured and modelled effective light absorption coefficients and the relative contribution of each photosynthetic pigment to the total light absorption under the culturing LEDs within T. erythraeum IMS101, when acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

Variables	Units	N2	NH4+	NO3-
aeffChl	m2 (g Chla)-1	9.9 (0.6)	7.7 (0.9)	8.1 (0.3)
aeffC	m2 (g C)-1	0.111 (0.013)	0.154 (0.020)	0.149 (0.006)
amodChl	m2 (g Chla)-1	10.0 (0.6)	7.8 (0.9)	8.3 (0.3)
amodC	m2 (g C)-1	0.112 (0.013)	0.156 (0.019)	0.154 (0.005)
Chla	%	35.66 (0.41)	36.74 (0.40)	39.18 (2.14)
PPC	%	30.64 (3.19)	27.48 (3.05)	27.44 (2.87)
PUB1	%	2.67 (1.36)[A]	5.04 (4.67)	10.11 (2.13)[B]
PUB2	%	1.34 (0.28)[B]	1.63 (1.04)	0.06 (0.06)[A]
PUBx	%	0	0	0
PUB4	%	0.02 (0.02)	0	0
PUB5a	%	0.42 (0.21)	0	0
PUB5b	%	0.24 (0.23)	0	0
PUB5d	%	0.05 (0.03)	0	0
PUBg	%	0.18 (0.18)	0	0
PUBj	%	0.19 (0.19)	0	0
PE1	%	8.10 (3.73)	7.51 (3.70)	3.19 (2.01)
PE2a	%	1.17 (0.75)	0	0
PE2b	%	1.02 (0.72)	0	0
PE3b	%	10.93 (2.28)	13.14 (3.60)	13.03 (0.92)
APC	%	5.45 (1.30)	5.17 (1.34)	5.97 (1.17)
PC1	%	0.94 (0.57)	0.19 (0.19)	0
PC2	%	2.22 (1.15)	3.08 (1.61)	1.02 (0.52)

Light absorption coefficients were spectrally corrected to the culture LEDs and were normalised to a chlorophyll a (m² g Chla^-1) and carbon (m² g C^-1) basis. Abbreviations; a_eff^Chl and a_eff^C are the measured Chla- and C-specific light absorption coefficients, while a_mod^Chl and a_mod^C are the modelled Chla- and C-specific light absorption coefficients. a_mod^Chl and a_mod^C were constructed from a range of pigment light absorption spectrums (λ = 400–700); comprising chlorophyll a (Chla), photoprotective carotenoids (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2). Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A].

In vivo light absorption spectra (Fig 1) exhibited peaks at ~ 440 nm (Chla), ~ 490–500 nm (phycourobilin; PUB), ~ 540 and 568 nm (phycoerythrin; PE), ~ 620 nm (phycocyanin; PC), ~ 640 nm (allophycocyanin; APC) and ~ 675 nm (Chla) (Table 3). Chlorophyll a and photoprotective carotenoids (PPC) dominated light absorption, together accounting for ~ 65% of the total. PUB1 and PUB2 were the only pigments to exhibit significant differences, where relative to the N₂ treatment, the contribution of PUB1 to the total light absorption increased by 7.4% whereas PUB2 decreased by 1.3% in the presence of NO₃^- (Table 3).

Fig 1. The mean (± S.E.) Chla (a-c) and C-specific (d-f) in vivo light absorption spectra for T. erythraeum IMS101 (n = 3).

Cultures were acclimated to three N-source treatments (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C). The solid black line is the measured light absorption spectra (grey area represents the S.E.) while the dashed line is the modelled light absorption spectra.

Light-dependence of O₂ exchange

The C-specific maximum rate (E_0m^C) and initial slope (α_g^C) of light-dependent gross photosynthesis increased with additional N-sources (i.e. NH₄⁺ and NO₃^-) and was highest for the NH₄⁺ treatment relative to the N₂ treatment (Table 4). There were also significant effects of additional N-sources on the Chla-specific maximum rate (E_0m^Chl) and initial slope of light-dependent gross photosynthesis (α_g^Chl) (S1 Table), however the effects were more pronounced when expressed as a C-specific rate, where E_0m^C increased by 143% from the N₂ to the NH₄⁺ treatment, while E_0m^Chl increased by only 36%.

Table 4. The parameters (± S.E.) of the C-specific light-response curves for gross and net photosynthetic O₂ evolution of T. erythraeum IMS101 (n = 3).

Parameters	Units	N2	NH4+	NO3-
Gross O2 evolution
E0mC	mmol O2 (g C)-1 h-1	6.05 (0.37)[A]	14.71 (1.20)[C]	10.98 (0.33)[B]
Ek	μmol photons m-2 s-1	238 (55)	227 (44)	255 (55)
αgC	μmol O2 (g C)-1 h-1 (μmol photons m-2 s-1)-1	27.9 (5.3)[A]	67.7 (8.2)[B]	49.8 (15.1)
ɸmgross	mol O2 (mol photons)-1	0.07 (0.01)[A]	0.12 (0.01)[B]	0.09 (0.03)
E0:Nfix	mol O2 (mol N2)-1	31 (4)[A]	289 (32)[B]	185 (57)[B]
Net Photosynthesis
PnetmC	mmol O2 (g C)-1 h-1	3.75 (0.27)[A]	11.48 (1.56)[B]	9.59 (0.37)[B]
Ek	μmol photons m-2 s-1	250 (69)	277 (8)	220 (37)
αnC	μmol O2 (g C)-1 h-1 (μmol photons m-2 s-1)-1	16.8 (3.4)[A]	41.5 (5.8)[B]	46.1 (8.0)[B]
RdC	mmol O2 (g C)-1 h-1	-1.63 (0.19)	-1.53 (0.28)	-1.16 (0.76)
ɸmnet	mol O2 (mol photons)-1	0.04 (0.01)[A]	0.08 (0.02)[B]	0.09 (0.01)[B]
Pnet:Nfix	mol O2 (mol N2)-1	18 (1)[A]	207 (29)[B]	163 (36)[B]
Gross (x) vs. Net (y)
slope	Dimensionless	0.60 (0.02)[A]	0.82 (0.03)[B]	0.83 (0.01)[B]

Abbreviations; E_0m^C, the C-specific maximum gross O₂ evolution rate; P_netm^C, the C-specific maximum net O₂ evolution rate; E_k, the light saturation parameter; α_g^C and α_n^C are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸ_mgross and ɸ_mnet are the maximum quantum efficiencies of gross and net O₂ evolution; R_d^C, the C-specific dark respiration rate; slope, the gradient of the regression between P_net^C and E₀^C; E₀:N_fix and P_net:N_fix, the ratio of gross and net photosynthesis to N₂ fixation, where rates of E₀ and P_net were calculated at 400 μmol photons m^-2 s^-1, matching to light intensity of the N₂ fixation incubations; slope, the gradient of the regression between P_net^C and E₀^C. The r² values of all curve fits were > 0.982. Letters in parenthesis indicate significant differences between CO₂ treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A].

The light saturation parameter (E_k = E_0m^C/α_g^C) of gross O₂ evolution did not vary significantly between N-source treatments (Table 4) and was due to covariation of α_g^C and E_0m^C. The maximum quantum efficiency of gross O₂ evolution (ɸ_mgross = α_g^C/a_eff^C) increased significantly by 76% from the N₂ to NH₄⁺ treatment (Table 4) and was due to the relatively constant a_eff^C and the significant increase in α_g^C.

Carbon-specific dark respiration rates (R_d^C) varied by ~ 24% and were slightly higher for the N₂ and NH₄⁺ treatments than the NO₃^- treatment (Table 4). Light-saturated net O₂ evolution rates (P_netm^C) approximately trebled and more than doubled from the N₂ treatment to the NH₄⁺ and NO₃^- treatments respectively (Table 4); with the initial slope (α_n^C) showing a similar pattern to P_netm^C. This increase in α_n^C for the NH₄⁺ and NO₃^- treatments resulted in the maximum quantum efficiency of net O₂ evolution (ɸ_mnet = α_n^C/a_eff^C) increasing significantly by 86% and 100% respectively, relative to the N₂ treatment (Table 4). The light saturation parameter (E_k = P_netm^C/α_g^C) for net O₂ evolution did not vary significantly between N-source treatments (Table 4).

The relationship between net and gross O₂ evolution was linear (Fig 2D–2F), with the slope increasing by approximately 40% when cultured in the presence of an additional N-source (Table 4). This linear relationship suggests that light-dependent O₂ consumption (U₀^C) was a constant proportion of gross O₂ evolution (E₀^C) and was independent of light intensity for all N-source treatments. Subtracting the slope from unity gave the ratio of light-driven U₀^C to E₀^C, which was significantly lower for the N₂ treatment.

Fig 2. The C-specific light response curves for gross O₂ evolution, O₂ consumption, net photosynthesis (n = 3) (a-c) and the relationship between gross and net O₂ evolution (d-f) for T. erythraeum IMS101.

Cultures were acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C). Chla-specific light response curves are shown in S4 Fig, while the light response curves for individual replicates are shown in S6–S8 Fig.

The ratio of gross photosynthesis (E₀) to N₂ fixation increased 9-fold and 6-fold for the NH₄⁺ and NO₃^- treatments relative to the N₂ treatment. In addition, the ratio of net photosynthesis (P_net) to N₂ fixation was 12-fold and 7-fold higher for the NH₄⁺ and NO₃^- treatments relative to the N₂ treatment (Table 4).

Light-dependence of PSII electron transport

The operating efficiency of PSII photochemistry (F_q'/F_mʹ) increased at low light intensities, reaching a maximum at ~ 110 to 130 μmol photons m^-2 s^-1, before decreasing significantly with increasing light intensity (Fig 3). The light saturation parameter (E_k) and the light at which ETR was maximal (E_opt) were significantly higher for the N₂ treatment than the NH₄⁺ treatment. Conversely, the light inhibition parameter (E_p), absorption cross-section of PSII photochemistry (σ_PII) and the time constant for the re-opening of a closed PSII reaction centre (τ_f) in the dark-adapted state were not significantly different between N-source treatments. Furthermore, both σ_PIIʹ and τ_fʹ exhibited no light-dependency, remaining relatively constant across the entire range of actinic light intensities (Fig 3, Table 5).

Fig 3. The operating efficiency of PSII photochemistry (F_qʹ/F_mʹ) (a-c), light absorption cross-section of PSII photochemistry (σ_PIIʹ) (d-f) and average time constant for the re-opening of a closed PSII reaction centres (τ_fʹ) (g-i) across a range of actinic light intensities for T. erythraeum IMS101 (n = 3).

Cultures were acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

Table 5. The parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeum IMS101 (n = 3).

Parameters	Units	N2	NH4+	NO3-
ETRmC	mmol e- (g C)-1 h-1	62.5 (16.7)	70.3 (14.4)	91.0 (4.9)
Ek	μmol photons m-2 s-1	465 (8)[B]	421 (3)[A]	447 (20)
αETRC	mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1	0.133 (0.033)	0.167 (0.033)	0.200 (0.003)
βETRC	mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1	5081 (55)[A]	5577 (55)[C]	5332 (14)[B]
Eopt	μmol photons m-2 s-1	1263 (22)[B]	1144 (3)[A]	1216 (54)
Ep	μmol photons m-2 s-1	0.99 (0.11)	0.67 (0.08)	0.83 (0.09)
Fv/Fm	Dimensionless	0.44 (0.01)	0.35 (0.05)	0.32 (0.01)
σPII	nm2 PSII-1	0.353 (0.009)	0.367 (0.003)	0.380 (0.032)
τf	s-1	489 (5)	494 (15)	631 (105)
Φem	mol e- (mol O2)-1	10.5 (1.1)[B]	5.7 (1.1)[A]	7.9 (0.5)
Φeα	mol e- (mol O2)-1	5.2 (0.8)	2.8 (0.2)	4.5 (1.0)

Abbreviations; ETR_m^C, the C-specific maximum electron transport rate; α_ETR^C, the C-specific initial slope of the electron transport rate light response curve; β_ETR^C, the C-specific light saturated slope of the electron transport rate light response curve; E_k, the light saturation parameter; E_opt, the light at which ETR is maximal; E_p, the light inhibition parameter; F_v/F_m, the maximum photochemical efficiency of PSII in the dark-adapted state; σ_PII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τ_f, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Φ_em, the light saturated ratio of PSII electron transport to gross O₂ evolution; Φ_eα, the light limited ratio of PSII electron transport to gross O₂ evolution. The r² values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A].

The light intensity at which ETR was maximal (E_opt) was significantly lower (by ~ 120 μmol photons m^-2 s^-1) for the NH₄⁺ treatment relative to the N₂ treatment (Fig 4). The Chla and C-specific maximum electron transport rate and initial slope (α_ETR) of the ETR-light curves were not significantly different between N-source treatments (Table 5, S2 Table). In contrast, the light-saturated photoinhibition slopes (β_ETR) were significantly different, with β increasing by 5% and 10% for the NO₃^- and NH₄⁺ treatments, relative to the N₂ treatment (Table 5).

Fig 4. Concurrent Chla (a-c) and C-specific (d-f) gross O₂ evolution rates and PSII electron transport rates (ETR) for T. erythraeum IMS101 (n = 3).

Cultures were acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

The ratio of PSII electron transport to gross O₂ evolution under light-limitation (Φ_eα) was ~ 4 and did not vary significantly between N-source treatments. Light saturated ratios (Φ_em) increased relative to Φ_eα for all N-source treatments, with the N₂ treatment being 46% and 35% higher than the NH₄⁺ and NO₃^- treatments, respectively (Table 5).

Discussion

Effect of acclimation to variation of N-sources on growth rates and elemental stoichiometry

Growth rates achieved under diazotrophic conditions were similar to most previous studies [23, 43–45], as was the increase in growth rate observed under non-diazotrophic conditions [23, 43], which we attribute to the lowered demand of NADPH and ATP for nitrogenase activity, where NADPH and ATP could be re-directed to CO₂ fixation and/or biosynthesis. Our data shows that at saturating light intensity, the energetic cost of diazotrophy constrains Trichodesmium growth by ~ 13%. However, in a natural system, potential changes to inorganic carbon chemistry (influencing the activity of the carbon concentrating mechanism (CCM)), temperature (influencing enzyme activity), or other key nutrients (i.e. Fe, P), all of which were controlled in our experiments, will almost certainly influence this estimate.

The decrease in C:N, C:P and N:P under non-diazotrophic conditions is consistent with previous findings [43]. The high C:N under diazotrophic conditions may be due to accumulation of stored glycogen, whereas the decrease in C:N under non-diazotrophic conditions is likely due to high cellular N concentrations, likely due to the luxury uptake of NH₄⁺ and NO₃^-, where surplus N is stored within cyanophycin granules [26]. Given the concurrent decrease in C:N, C:P and N:P under non-diazotrophic conditions, it is likely that utilising NH₄⁺ or NO₃^- as a N-source enables Trichodesmium cells of low carbon biomass to maintain a Chla concentration comparable to diazotrophic conditions. This is supported by previous observation made by Eichner et al. [43] and is also reflected by the higher Chla:C yet comparable Chla:N ratios for NH₄⁺ or NO₃^- treatments.

Growing evidence points towards nitrogenase being expressed in subsets of cells within filaments, called diazocytes [21, 25]. To date, no translocation transport mechanisms for N compounds have been observed in Trichodesmium, leading to suggestions that diazocytes release N into the external medium for use by neighbouring cells [25, 46]. This is partially supported by observations of Trichodesmium exhibiting a high capacity for NH₄⁺ uptake during active N₂ fixation [17, 18]. While such mechanisms may exist, we did not observe significant concentrations of dissolved inorganic NO₃^- or NH₄⁺ in the medium of our control treatment.

Effect of acclimation to different N-sources on gross photosynthesis

We show an effect of N-source on C-specific light saturated gross O₂ evolution rates. The more than two-fold increase in the maximum O₂ evolution rate and initial slope when T. erythraeum IMS101 was grown on NH₄⁺ or NO₃^- than when growing diazotrophically was largely due to differences in the ratio of Chla:C as chlorophyll a-specific photosynthetic parameters varied by only 36% between N₂ and NH₄⁺ treatments.

The increase of C-specific gross O₂ evolution rates when Trichodesmium is supplied with NH₄⁺ or NO₃^- may be due to an increase in the maximum rate of CO₂ fixation and/or to an increase in PSII concentration. Previous studies report high PSI:PSII ratios under diazotrophic conditions (ranging between 1.3 to 4) [47–51], which would allow cyclic photophosphorylation in diazocytes to provide most of the ATP required for N₂ fixation, with glycolysis and the Kreb’s cycle providing the required reducing equivalent. It may be that under non-diazotrophic conditions and with lower nitrogenase activity, Trichodesmium enhances linear electron transport to increase NADPH production; a pathway that generates more evolved O₂.

Effect of acclimation to different N-sources on N₂ fixation

Nitrogenase activity declined significantly by 81–84% when Trichodesmium was cultured in the presence of an additional N-source. Despite being cultured under N-replete concentrations, Trichodesmium cells in the NH₄⁺ and NO₃^- treatments exhibited a baseline rate of N₂ fixation. Similarly, Milligan et al. [52] reported a ~ 85% decrease when Trichodesmium was cultured in 100 μM of NO₃^- for 2 weeks and Holl and Montoya [44] reported a 66% decrease when cultured in 20 μM of NO₃^-, accrediting 8% of total N assimilation to diazotrophy despite the presence of additional N-sources. Maintaining the capability to perform N₂ fixation under non-diazotrophic conditions, albeit at a reduced rate, could reflect Trichodesmium’s natural environment and act a potential safeguard mechanism to variable light and nutrient regimes.

Noting that 16 moles of ATP are consumed per mole of N₂ fixed (Eq 1) and that 2.56 moles of ATP can be produced per mole of O₂ evolved by photophosphorylation linked LPET [53], we calculated that T. erythraeum IMS101 may use 20% of the ATP that could be generated from gross O₂ evolution to support the observed N₂ fixation rate during diazotrophic growth:

$$\frac{N_{fix}}{E_0}=\frac{{1molN}_2}{{31molO}_2}\bullet \frac{{1molO}_2}{2.54molATP}\bullet \frac{16molATP}{{1molN}_2}=0.2$$ (24)

This proportion decreases to 2% and 4% for the NO₃^- and NH₄⁺ treatments, respectively, where the ratio of E₀:N_fix increases to 289 for the NO₃^- treatment and 185 in the NH₄⁺ treatment, versus 31 in the N₂ treatment (Table 4).

Studies on natural populations of Trichodesmium spp. have shown that the addition of NO₃^- (100 μM) in the morning can cause a gradual decrease of N₂ fixation over the photic period [22]. Further studies have also shown that addition of glutamine (10 μM) immediately decreases N₂ fixation rates, indicating a direct effect on enzyme activity as opposed to enzyme synthesis [54]. These observations have been accredited to accumulation of N-containing metabolites acting as potential inhibitors to the specific activity rather than abundance of nitrogenase [22, 54].

It is well known that intracellular nitrogen pools have a role in regulating nitrogenase activity in diazotrophs [55, 56]. Dinitrogenase reductase catalyses the reduction of N₂ to NH₄⁺, which is assimilated into glutamine (gln) and then into glutamate (glu) via the glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) pathway [54]. The intracellular pools of NH₄⁺, glu and gln have been identified as important feedback regulators of N uptake and metabolism, with GS activity in Trichodesmium being sensitive to both intra- and extracellular N concentrations [55]. It could be hypothesised that the activity of nitrogenase is influenced by internally recycled N (e.g. NH₄⁺ and gln), while the synthesis of nitrogenase is influenced by newly assimilated N (e.g. NO₃^-).

Effect of acclimation to different N-sources on light-stimulated O₂ consumption and the relationship between net and gross O₂ evolution

Net photosynthesis was significantly lower for the N₂ treatment than for the NH₄⁺ and NO₃^- treatments. Despite slight variations in E₀^C, the difference in net photosynthesis was principally driven by O₂ consumption. Approximately 68%, 32% and 29% (N₂, NH₄⁺ and NO₃^-, respectively) of E₀^C was consumed by O₂ consuming processes, which is comparable to previous observations [43, 52].

Several processes demand ATP in excess of the ATP:NADPH produced through linear photophosphorylation; two most notably being N₂ fixation and the operation of the CCM [57]. In this study, the carbon chemistry of all cultures was closely regulated to ensure that variation in O₂ consumption and net photosynthesis was due to the N-source treatments only. Linearity between gross O₂ evolution (E₀) and O₂ consumption was observed across all N-source treatments, suggesting that light-dependent O₂ consumption is linked to balancing ATP to NADPH production, as opposed to serving as a mechanism to dissipate excitation energy.

Diazotrophic cells consume more O₂ per evolved O₂ across the entire range of actinic light intensities than the NH₄⁺ and NO₃^- treatments. This suggests a higher rate of water-water cycling due to either Mehler activity or operation of plastoquinone terminal oxidase when N₂ is being fixed. To maintain a sufficient supply of ATP relative to NADPH, Trichodesmium may utilise pseudocyclic photophosphorylation linked to the Mehler reaction to augment the ATP generated by linear electron transfer from water to NADP⁺ in addition to ATP produced by cyclic electron flow around PSI.

Measurements of O₂ evolution, ETR and N₂ fixation were all made at one time of day (4 to 6 hours into the photo-phase of a 12:12 L:D cycle) and as such cannot be extrapolated to a diel response given the reports of temporal separation of photosynthesis and N₂ fixation in Trichodesmium [21].

Effect of acclimation to different N-sources on electron transport rates and photophysiology

Like Eichner et al. [43], we observed a negligible effect of N-source on many photo-physiological parameters, including F_qʹ/F_mʹ, σ_PII and τ_f. Trichodesmium exhibited a light response typical for most cyanobacteria, where the dark-adapted photochemical yield is significantly affected by respiratory electron flow [58]. This results from a proportion of PSII reaction centres remaining in a closed state despite being in the dark and is imposed by a reduction in the plastoquinone (PQ) pool, which prevents the oxidation of Q_A^-. Moving from darkness to a low light intensity increases the electron flux through PSI, alleviates the bottleneck of electron transport through the Cyt b6f complex, thereby increasing F_qʹ/F_mʹ and decreasing the re-oxidation time of Q_A^-. Addition factors such as higher downregulation under dark-adapted conditions may also contribute to the increase in F_qʹ/F_mʹ under low light intensities.

Ratio of electron transport to gross O₂ evolution

Electrons are transferred from PSII (where O₂ is evolved) to an intermediate plastoquinone pool and eventually to ferredoxin to produce NADPH [59]. A minimum of four moles of electrons are transported through PSII for each mole of O₂ evolved at PSII. Most higher plants exhibit a linear correlation between gross O₂ evolution and electron transport rate [60]. In microalgae, this relationship is often ambiguous, especially at high light intensities where the relationship can become non-linear [61, 62].

Here we show that at low light intensities, the ratio of PSII electron transport to gross O₂ evolution (Φ_eα) is close to a 4:1 ratio for all N-sources treatments. However, when light intensities exceed 150 μmol photons m^-2 s^-1, Φ_e declines as ETR saturates at a higher light intensity (~ 900 μmol photons m^-2 s^-1) than E₀ (~ 400 μmol photons m^-2 s^-1). Similar responses have been reported for diatoms [63], microalgae [64] and the Baltic cyanobacteria, Nostoc [65]. Few studies have measured O₂ production rates in Trichodesmium [47, 66] and to our knowledge none have reported concurrent PSII electron transport rates.

Interestingly, we calculated a higher Φ_e for the N₂ cultures than for the NH₄⁺ and NO₃^- cultures, irrespective of using the light-limited or -saturated rates. This may be due to overestimating the proportion of light absorbed by PSII in the non-diazotrophic growth conditions (i.e. NH₄⁺ and NO₃^-) relative to the diazotrophic condition. Here we assumed that 50% of absorbed light was directed to PSII reaction centres and 50% to PSI reaction centres (i.e. FAQ_PII of 0.5). It’s likely that FAQ_PII was overestimated for diazotrophic treatment (i.e. N₂) which may have had a higher ratio of PSI:PSII to support significant rates of cyclic photophosphorylation. In addition, non-diazotrophic cells may undergo more pronounced state transitions with phycobilin proteins being redistributed between PSII and PSI. Finally, a Φ_e > 4 could be accredited to cyclic electron flow around PSII, which may act a mechanism to dissipate excess excitation energy under high light [67].

Implications for future oligotrophic oceans

In N-limited regions of the oligotrophic open ocean, diazotrophy provides a competitive advantage by allowing cells to access N₂ as an N-source against faster growing phytoplankton that rely on fixed N. Current ocean models predict a poleward shift in the 20 °C isotherm which could extend Trichodesmium’s niche into higher latitudes. On a global scale, this niche expansion is driven by increased SSTs; however, on regional scales persistence in an area may be dictated by Trichodesmium’s response to fluctuating nutrient regimes.

At the surface in oligotrophic waters, Trichodesmium is unlikely to encounter NO₂^-, NO₃^- or NH₄⁺ concentrations in excess of 0.1 μM [68], except during mixing events. While Trichodesmium is commonly observed in the upper meters of the water column [69], observations have been recorded down to 200 m depth [70]. Thus, Trichodesmium colonies and free trichomes are able to migrate to the nutricline [30, 31]. Such vertical migration has been suggested to allow luxury uptake of phosphates before colonies return to the surface. In addition to encountering phosphates, Trichodesmium will also encounter high concentrations of NO₃^- in the nutricline. As such, NO₃^- uptake is likely at these greater depths or at the surface after a mixing event.

Mulholland et al. [17] reported significant NO₃^- uptake rates with the addition of 1 μM NO₃^- to the growth media. Furthermore, Karl et al. [30] showed that concentrations of dissolved NH₄⁺ reached 1.5 μM L^-1 and dissolved organic N (DON) reaching 13 μM L^-1 during a natural bloom of Trichodesmium spp. in the North Pacific gyre. These concentrations are far greater than typical oceanic N pools and could therefore be high enough to inhibit N₂ fixation rates [44]. It’s therefore possible that Trichodesmium colonies at depth may be utilising more combined N-sources than the blooms frequently measured on the surface. The energy and reductant conserved through utilising additional N-sources could significantly enhance Trichodesmium’s productivity and growth which could have major implications for biogeochemical cycles.

Our results indicate the need to seek more information on the potential for natural populations of Trichodesmium to uptake fixed N-sources (e.g. NO₃^-, NH₄⁺, labile dissolved organic nitrogen (DON)) at concentrations that migrating colonies or trichomes experience in the nutricline or that are encountered transiently after deep mixing events. The potential significance of Trichodesmium assimilating fixed N is indicated by a modelling study by McGillicuddy [33] which concluded that to obtain realistic simulations of biomass and export production Trichodesmium populations in the North Atlantic must utilise fixed N. Specifically, this study indicated that 15–20% of the N quota of Trichodesmium could be due to uptake of NO₃^- and NH₄⁺. Furthermore, although uptake of NO₃^-, NH₄⁺ or DON will decrease N₂ fixation rates in the short-term, as these N-sources are depleted over longer time periods, the increase in Trichodesmium biomass may lead to increased N₂ fixation and greater competition for other nutrients including Fe and P.

Supporting information

S1 Fig. The relative fluorescence excitation spectra of T. erythraeum IMS101 (Bold solid line) and the relative emission spectra of the Iso Light 400 LED (white) block used for O₂ evolution incubations (Solid line), FRRf LED (blue) used for the saturating flashlets (Long-Dashed line), FastAct LED (white) used for the actinic light source (Short-dashed line) and the culturing LED (white) (Dotted line).

(A) The fluorescence excitation was measured on a 2 mL concentrated sample treated with 20 μM DCMU (final concentration) [71]. Trichodesmium cells were acclimated to 150 μmol photons m^-2 s^-1 on a 14:10 light:dark cycle, 26 °C and ambient CO₂. The sample was measured using a FluorWin fluorometer scanning between 400 to 715 nm at a 1 nm resolution, with the monochromator on the detector set to 730 nm emission [72]. Spectral correction factors were calculated using the FastPro8. (B) An example of an in vivo light absorption spectra of T. erythraeum IMS101 when spectrally corrected to the Culture, MIMS or FRRf LED spectra.

(TIF)

S2 Fig. Reconstructed light absorption spectra of eighteen key photosynthetic pigments present within T. erythraeum IMS101.

(A) The light absorption spectra of chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycoerythrin (PE), plastocyanin (PC) and alloplastocyanin (APC) pigments. (B) The light absorption spectra of phycourobilin (PUB) pigments. Each pigment spectra was normalised to the maximum peak (λ = 400–700 nm).

(TIF)

S3 Fig. Inorganic carbon chemistry (Ci) of T. erythraeum IMS101 cultures, measured at 2-hour intervals over the light period.

The pH and TCO₂ was measured directly, while the pCO₂ concentrations were calculated via CO2SYS using the same constants as described in Boatman et al. [45] and S1 File.

(TIF)

S4 Fig. Chla-specific light response curves for gross O₂ evolution, O₂ consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O₂ evolution (D-F) for T. erythraeum IMS101.

Cultures were acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

(TIF)

S5 Fig. Percentage of the modelled in vivo light absorption (a_mod) associated to each photosynthetic pigment (λ = 400–700) for T. erythraeum IMS101.

Cultures were acclimated to three N-sources (N₂, NH₄⁺ and NO₃^-), at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C). Pigments include chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2).

(TIF)

S6 Fig. Chla and C-specific light response curves for gross O₂ evolution, O₂ consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O₂ evolution (D-F) for T. erythraeum IMS101.

Cultures were acclimated to N₂-only, at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

(TIF)

S7 Fig. Chla and C-specific light response curves for gross O₂ evolution, O₂ consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O₂ evolution (D-F) for T. erythraeum IMS101.

Cultures were acclimated to a replete NH₄⁺ concentration, at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

(TIF)

S8 Fig. Chla and C-specific light response curves for gross O₂ evolution, O₂ consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O₂ evolution (D-F) for T. erythraeum IMS101.

Cultures were acclimated to a replete NO₃^- concentration, at a target CO₂ concentration (380 μatm), saturating light intensity (400 μmol photons m^-2 s^-1) and optimal temperature (26 °C).

(TIF)

S1 Table. Physiological parameters (± S.E.) of the Chla-specific light-response curves for gross and net photosynthetic O₂ evolution of T. erythraeum IMS101 (n = 3).

Abbreviations; E_0m^Chl, the Chla -specific maximum gross O₂ evolution rate; P_m^Chl, the Chla -specific maximum net O₂ evolution rate; α_g^Chl and α_n^Chl are the Chla -specific initial slopes the light response curve for net and gross photosynthesis; R_d^Chl, the Chla-specific dark respiration rate. The r² values of all curve fits were > 0.982. Letters in parenthesis indicate significant differences between CO₂ treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A].

(PDF)

S2 Table. Physiological parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeum IMS101 (n = 3).

Abbreviations; ETR_m^Chl, the Chla-specific maximum electron transport rate; α_ETR^Chl, the Chla-specific initial slope of the electron transport rate light response curve; β_ETR^Chl, the Chla-specific light saturated slope of the electron transport rate light response curve. The r² values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A].

(PDF)

S1 File. Calculation of inorganic carbon speciation.

(PDF)

S2 File. Calculation of dissolved inorganic N concentration.

(PDF)

S3 File. Measuring O₂ production and consumption.

(PDF)

S4 File. MIMS sample preparation.

(PDF)

S5 File. Elemental stoichiometry.

(PDF)

S6 File. Spectrophotometric chlorophyll a analysis.

(PDF)

S7 File. Spectrally corrected in vivo light absorption.

(PDF)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Tobias Boatman was supported by a UK Natural Environment Research Council PhD studentship (NE/J500379/1 DTB). Funding obtained by RJG and TL.