The physiological cost of diazotrophy for Trichodesmium erythraeum IMS101.

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. n>an class="Chemical">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].

pan class="Chemical">Previous studies have highlighted pan class="Species">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

pan class="Chemical">Diazotrophic cyanobacteria are able to meet their daily pan class="Chemical">nitrogen quota by fixing dinitrogen (N2).

While pan class="Chemical">N2 fixation is an extremely energy demanding process, n>an class="Species">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 pan class="Chemical">diazotrophic cyanobacteria spp., n>an class="Species">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).

For cyanobacteria, pan class="Chemical">nitrate reductase is located in the cytosol and uses pan class="Chemical">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;

Amino acids are synthesised from pan class="Chemical">ammonia (pan class="Chemical">NH3) via the GS-GOGAT pathway. The initial GS pathway requires ATP and glutamate as a substrate; where glutamine is subsequently transformed to 2-oxoglutarate and reduced using NADPH, forming two moles of glutamate.

Thus, for every mole of pan class="Chemical">glutamate produced, one mole each of n>an class="Chemical">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 pan class="Chemical">water stratification and decreasing vertical mixing [29], potentially increasing the area of N-limited oceans. Whilst pan class="Disease">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>an class="Chemical">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

pan class="Species">T. erythraeum IMS101 was semi-continuously cultured to achieve fully acclimated balanced growth at three pan class="Chemical">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 pan class="Chemical">CO2 and light intensity for ~ 4 months (~ 60 generations) under pan class="Chemical">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).

pan class="Species">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) measured between 09:00 to 10:30 on dark-adapn>ted 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 versus time and the ratio of live cell to n>an class="Chemical">acetone extracted (method detailed below) baseline fluorescence (F) 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 pan class="Chemical">inorganic pan class="Chemical">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 pan class="Chemical">O2 production and consumption were measured with a membrane inlet mass spn>ectrometer (MIMS), using an n>an class="Chemical">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 16pan class="Chemical">O2 and pan class="Chemical">18O2 and thus O2 consumption (Uo) and O2 evolution (Eo) were calculated using the following equations [36]; where Uo 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 Eo 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).

pan class="Chemical">Photosynthesis-light (pan class="Chemical">P-E) curves for gross (E0Chl(C)) and net photosynthesis (PnetChl(C) = E0Chl(C)-U0Chl(C)) were fitted to the equations from Platt and Jassby [37]; where E0mChl(C) and PnetmChl(C) are the maximum gross and net O2 evolution rates; αg Chl(C) and αnChl(C) are the initial light-limited slopes for gross and net photosynthesis; Rd 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 (ɸmgross) and net (ɸmnet) pan class="Chemical">O2 evolution was calculated as follows; where the C-specific initial slope for gross (αgC) or net (αnC) n>an class="Chemical">O2 evolution was divided by the C-specific, effective light absorption coefficient (aeffC).

Measuring nitrogenase activity by acetylene reduction

pan class="Chemical">Acetylene reduction rates were measured using gas chromatography (ATI Unicam 610 series). Gaseous sampn>les were injected into the GC column head (60 °C), carried via n>an class="Chemical">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; where (ΔC2H2) is the ethylene production rate (μmol C2H4 h-1), C2H2(T0) and C2H2 (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).

pan class="Chemical">N2 fixation rates were calculated to a Chla (μmol pan class="Chemical">N2 (mg Chla)-1 h-1) and total carbon (μmol N2 (mg C)-1 h-1) basis; 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 (σn>an class="Chemical">PIIʹ), the average time constant for the re-opening of a closed PSII reaction centre (τfʹ) and the operating efficiency of PSII photochemistry (Fʹ/Fʹ); where Fʹ is the maximum fluorescence in the light-adapted state and Fʹ is the steady-state fluorescence at any point.

pan class="Chemical">Photosystem II (pan class="Chemical">PSII) electron transport rates were normalised to a Chla (mol e- (g Chla)-1 h-1) and total pan class="Chemical">carbon (mol e- (g C)-1 h-1) basis; where Fʹ/Fʹ is the operating efficiency of PSII photochemistry; E is the light intensity (mol photons m-2 s-1), aChl(C) is the Chla-specific (C-specific) effective light absorption (m2 g-1 Chla and m2 g-1 C, respectively), FAQPII 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).

pan class="Chemical">ETR curves were modelled using a pan class="Chemical">P-E equation [37], performed on each individual replicate using a Marquardt–Levenberg least squares algorithm to generate the best fit (R2 > 0.993); where ETRmʹ 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); αETR 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); βETR 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 pan class="Chemical">PSII electron transport rate in the presence of photoinhibition (ETRm), light intensity at which ETR is maximal (Eopt), the light-saturation parameter (Ek) and the light inhibition parameter (Ep) were calculated from the fitted parameters as follows:

The ratio of pan class="Chemical">PSII electron transport to gross n>an class="Chemical">O2 evolution (E0) under light-limitation (Φeα) and light-saturation (Φem) were calculated as follow;

Cellular elemental composition and light absorption

Samples for determining particulate organic carbon (n>an class="Chemical">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 (n>an class="Chemical">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; where aChlmod is the modelled in vivo light absorption at a specific wavelength (λ = 400–700 nm); βi is the contribution of each pigment to aChlmod 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 (aChlmod (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 pan class="Species">T. erythraeum IMS101 was 0.34 d-1 when grown on pan class="Chemical">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].

Abbreviations; C:N, C:pan class="Chemical">P and N:pan class="Chemical">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 pan class="Chemical">inorganic pan class="Chemical">carbon concentration, pH and alkalinity (AT) 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 efpan class="Chemical">fective light absorption coefficients were not significantly difn>an class="Chemical">ferent 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; aeffChl and aeffC are the measured Chla- and C-specific light absorption coefficients, while amodChl and amodC are the modelled Chla- and C-specific light absorption coefficients. amodChl and amodC 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].

Light absorption coefficients were spectrally corrected to the culture LEDs and were normalised to a chlorophyll a (m2 g Chla-1) and n>an class="Chemical">carbon (m2 g C-1) basis. Abbreviations; aeffChl and aeffC are the measured Chla- and C-specific light absorption coefficients, while amodChl and amodC are the modelled Chla- and C-specific light absorption coefficients. amodChl and amodC 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.

Cultures were acclimated to three pan class="Chemical">N-source treatments (pan class="Chemical">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 (E0mC) and initial slope (αgC) of light-dependent gross photosynthesis increased with additional N-sources (i.e. n>an class="Chemical">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 (E0mChl) and initial slope of light-dependent gross photosynthesis (αgChl) (S1 Table), however the effects were more pronounced when expressed as a C-specific rate, where E0mC increased by 143% from the N2 to the NH4+ treatment, while E0mChl 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; E0mC, the C-specific maximum gross O2 evolution rate; PnetmC, the C-specific maximum net O2 evolution rate; Ek, the light saturation parameter; αgC and αnC are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸmgross and ɸmnet are the maximum quantum efficiencies of gross and net O2 evolution; RdC, the C-specific dark respiration rate; slope, the gradient of the regression between PnetC and E0C; E0:Nfix and Pnet:Nfix, the ratio of gross and net photosynthesis to N2 fixation, where rates of E0 and Pnet 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 PnetC and E0C. 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].

Abbreviations; E0mC, the C-specific maximum gross pan class="Chemical">O2 evolution rate; class="Chemical">n>an class="Chemical">PnetmC, the C-specific maximum net O2 evolution rate; Ek, the light saturation parameter; αgC and αnC are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸmgross and ɸmnet are the maximum quantum efficiencies of gross and net O2 evolution; RdC, the C-specific dark respiration rate; slope, the gradient of the regression between PnetC and E0C; E0:Nfix and Pnet:Nfix, the ratio of gross and net photosynthesis to N2 fixation, where rates of E0 and Pnet 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 PnetC and E0C. 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 (Ek = E0mC/αgC) of gross pan class="Chemical">O2 evolution did not vary significantly between pan class="Chemical">N-source treatments (Table 4) and was due to covariation of αgC and E0mC. The maximum quantum efficiency of gross O2 evolution (ɸmgross = αgC/aeffC) increased significantly by 76% from the N2 to NH4+ treatment (Table 4) and was due to the relatively constant aeffC and the significant increase in αgC.

pan class="Chemical">Carbon-specific dark respn>iration rates (RdC) varied by ~ 24% and were slightly higher for the n>an class="Chemical">N2 and NH4+ treatments than the NO3- treatment (Table 4). Light-saturated net O2 evolution rates (PnetmC) approximately trebled and more than doubled from the N2 treatment to the NH4+ and NO3- treatments respectively (Table 4); with the initial slope (αnC) showing a similar pattern to PnetmC. This increase in αnC for the NH4+ and NO3- treatments resulted in the maximum quantum efficiency of net O2 evolution (ɸmnet = αnC/aeffC) increasing significantly by 86% and 100% respectively, relative to the N2 treatment (Table 4). The light saturation parameter (Ek = PnetmC/αgC) for net O2 evolution did not vary significantly between N-source treatments (Table 4).

The relationship between net and gross pan class="Chemical">O2 evolution was linear (Fig 2D–2F), with the slope increasing by approximately 40% when cultured in the presence of an additional n>an class="Chemical">N-source (Table 4). This linear relationship suggests that light-dependent O2 consumption (U0C) was a constant proportion of gross O2 evolution (E0C) and was independent of light intensity for all N-source treatments. Subtracting the slope from unity gave the ratio of light-driven U0C to E0C, 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.

Cultures were acclimated to three pan class="Chemical">N-sources (pan class="Chemical">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 pan class="Chemical">N2 fixation increased 9-fold and 6-fold for the pan class="Chemical">NH4+ and NO3- treatments relative to the N2 treatment. In addition, the ratio of net photosynthesis (Pnet) 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 pan class="Chemical">PSII photochemistry (F'/Fʹ) 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 (Ek) and the light at which n>an class="Chemical">ETR was maximal (Eopt) were significantly higher for the N2 treatment than the NH4+ treatment. Conversely, the light inhibition parameter (Ep), absorption cross-section of PSII photochemistry (σPII) and the time constant for the re-opening of a closed PSII reaction centre (τf) in the dark-adapted state were not significantly different between N-source treatments. Furthermore, both σPIIʹ and τfʹ 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 (Fʹ/Fʹ) (a-c), light absorption cross-section of PSII photochemistry (σPIIʹ) (d-f) and average time constant for the re-opening of a closed PSII reaction centres (τfʹ) (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; ETRmC, the C-specific maximum electron transport rate; αETRC, the C-specific initial slope of the electron transport rate light response curve; βETRC, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; F/F, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Φem, the light saturated ratio of PSII electron transport to gross O2 evolution; Φeα, 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].

Cultures were acclimated to three pan class="Chemical">N-sources (pan class="Chemical">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).

Abbreviations; pan class="Chemical">ETRmC, the C-specific maximum electron transpn>ort rate; αn>an class="Chemical">ETRC, the C-specific initial slope of the electron transport rate light response curve; βETRC, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; F/F, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Φem, the light saturated ratio of PSII electron transport to gross O2 evolution; Φeα, 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 pan class="Chemical">ETR was maximal (Eopt) was significantly lower (by ~ 120 μmol photons m-2 s-1) for the n>an class="Chemical">NH4+ treatment relative to the N2 treatment (Fig 4). The Chla and C-specific maximum electron transport rate and initial slope (αETR) of the ETR-light curves were not significantly different between N-source treatments (Table 5, S2 Table). In contrast, the light-saturated photoinhibition slopes (βETR) 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).

Cultures were acclimated to three pan class="Chemical">N-sources (pan class="Chemical">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 pan class="Chemical">PSII electron transport to gross n>an class="Chemical">O2 evolution under light-limitation (Φeα) was ~ 4 and did not vary significantly between N-source treatments. Light saturated ratios (Φem) increased relative to Φeα 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 pan class="Chemical">diazotrophic conditions were similar to most previous studies [23, 43–45], as was the increase in growth rate observed under non-n>an class="Chemical">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:pan class="Chemical">P and N:pan class="Chemical">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 pan class="Chemical">nitrogenase being expressed in subsets of cells within filaments, called diazocytes [21, 25]. To date, no translocation transport mechanisms for N compn>ounds have been observed in n>an class="Species">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 efpan class="Chemical">fect of pan class="Chemical">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 pan class="Chemical">O2 evolution rates when class="Chemical">n>an class="Species">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

pan class="Chemical">Nitrogenase activity declined significantly by 81–84% when pan class="Species">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 pan class="Chemical">ATP are consumed per mole of n>an class="Chemical">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:

This proportion decreases to 2% and 4% for the NO3- and n>an class="Chemical">NH4+ treatments, respectively, where the ratio of E0:Nfix 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 class="Chemical">n>an class="Chemical">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 pan class="Chemical">nitrogen pools have a role in regulating n>an class="Chemical">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 pan class="Chemical">N2 treatment than for the class="Chemical">n>an class="Chemical">NH4+ and NO3- treatments. Despite slight variations in E0C, the difference in net photosynthesis was principally driven by O2 consumption. Approximately 68%, 32% and 29% (N2, NH4+ and NO3-, respectively) of E0C was consumed by O2 consuming processes, which is comparable to previous observations [43, 52].

Several processes demand pan class="Chemical">ATP in excess of the class="Chemical">n>an class="Chemical">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.

pan class="Chemical">Diazotrophic cells consume more pan class="Chemical">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 pan class="Chemical">O2 evolution, pan class="Chemical">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 efpan class="Chemical">fect of pan class="Chemical">N-source on many photo-physiological parameters, including Fʹ/Fʹ, σPII and τf. 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 QA-. 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 Fʹ/Fʹ and decreasing the re-oxidation time of QA-. Addition factors such as higher downregulation under dark-adapted conditions may also contribute to the increase in Fʹ/Fʹ under low light intensities.

Ratio of electron transport to gross O2 evolution

Electrons are transpan class="Chemical">ferred from pan class="Chemical">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 pan class="Chemical">PSII electron transport to gross n>an class="Chemical">O2 evolution (Φeα) is close to a 4:1 ratio for all N-sources treatments. However, when light intensities exceed 150 μmol photons m-2 s-1, Φe 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 Φe for the pan class="Chemical">N2 cultures than for the pan class="Chemical">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. FAQPII of 0.5). It’s likely that FAQPII 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 Φe > 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 pan class="Chemical">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 pan class="Chemical">waters, pan class="Species">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 pan class="Chemical">NO3- uptake rates with the addition of 1 μM pan class="Chemical">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>an class="Chemical">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.

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]. pan class="Species">Trichodesmium cells were acclimated to 150 μmol photons m-2 s-1 on a 14:10 light:dark cycle, 26 °C and ambient pan class="Chemical">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)

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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 (pan class="Chemical">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).

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

The pH and Tpan class="Chemical">CO2 was measured directly, while the pan class="Chemical">pCO2 concentrations were calculated via CO2SYS using the same constants as described in Boatman et al. [45] and S1 File.

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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 pan class="Chemical">N-sources (pan class="Chemical">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).

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

Cultures were acclimated to three pan class="Chemical">N-sources (pan class="Chemical">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).

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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 pan class="Chemical">N2-only, at a target pan class="Chemical">CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 °C).

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

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

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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; E0mChl, the Chla -specific maximum gross pan class="Chemical">O2 evolution rate; class="Chemical">n>an class="Chemical">PmChl, the Chla -specific maximum net O2 evolution rate; αgChl and αnChl are the Chla -specific initial slopes the light response curve for net and gross photosynthesis; RdChl, 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].

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

Abbreviations; pan class="Chemical">ETRmChl, the Chla-specific maximum electron transpn>ort rate; αn>an class="Chemical">ETRChl, the Chla-specific initial slope of the electron transport rate light response curve; βETRChl, 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].

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Calculation of inorganic carbon speciation.

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Calculation of dissolved inorganic N concentration.

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Measuring O2 production and consumption.

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MIMS sample preparation.

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Elemental stoichiometry.

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Spectrophotometric chlorophyll a analysis.

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Spectrally corrected in vivo light absorption.

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