Two N₂-fixing cyanobacteria, T. erythraeum IMS101 and C. watsonii WH8501, along with one non- N₂-fixing pico-cyanobacterium, Synechococcus sp. WH8102, were cultured. T. erythraeum and C. watsonii were grown in Aquil-tricho medium (Hong et al., 2017) prepared with 0.22 µm-filtered and microwave-sterilized oligotrophic western North Pacific surface water. The medium was enriched with chelexed and filter-sterilized NaH₂PO₄, and supplied with filter-sterilized vitamins and trace metals, buffered with 5 µM EDTA. Synechococcus was also grown in Aquil-tricho medium but was enriched with 100 µM NaNO₃.

All algae were pre-adapted to a light intensity of 200 µE m^-2 s^-1 by semi-continuous culturing for more than six months. The light level was monitored using a spherical light meter (QSL-2100, Biospherical Instruments Inc., San Diego CA, USA). The light intensity measured using the spherical light meter was approximately 2.5 times greater than that measured using a flat light meter (~80 µE m^-2 s^-1). Cultures were maintained in the exponential growth stage at 27°C with a 14:10 h light−dark cycle in an algal growth chamber (AGC-850, Firstek Corp, China) before starting the co-culture. Strict sterile techniques were applied for culturing and experimental manipulations.

Exponentially growing Synechococcus was transferred to an inorganic-N-free Aquil-tricho medium for a 2-day N-starvation acclimation, during which cell growth ceased ( Supplementary Figure S1 ). The N-starved Synechococcus was then transferred separately into exponentially growing T. erythraeum and C. watsonii cultures, and co-cultured under the same conditions as the monoculture of T. erythraeum and C. watsonii. To achieve the exponential growth of diazotrophs and Synechococcus in the co-culture systems, inoculation ratios were optimized in the preliminary experiments. The optimized inoculation ratios were Synechococcus: T. erythraeum = ~5−8:1 (cell number: cell number), and Synechococcus: C. watsonii = 0.003:1 (cell number: cell number), respectively, which ensured a similar growth rate of Synechococcus in both co-cultures ( Table 1 ). To minimize the impact of the inoculation process, the volume of the Synechococcus culture transferred was kept to less than 7% of the T. erythraeum and C. watsonii culture volumes. In the co-culture system, N fixed by diazotrophs was the sole N source supporting Synechococcus growth. Both the diazotrophs and Synechococcus in the co-cultures continued exponential growth for at least 3 days (as shown in Figure 1 ), after which an aliquots of the co-cultures were spiked with ¹⁵N₂ gas (98.9 atom%, Cambridge Isotope Laboratories, Lot #: I-21065/AR0664758) and incubated for another 24 h to determine the total N₂ fixation rate of diazotrophs, as well as the DDN release to the dissolved phase and DDN transfer to Synechococcus.

To explore the impact of light intensity on DDN release and transfer, T. erythraeum and Synechococcus were adapted to a lower light intensity of 70 µE m^-2 s^-1, and a higher light intensity of 750 µE m^-2 s^-1 for at least 3 months. Subsequently co-cultures of T. erythraeum and Synechococcus were established as described above. Additionally, to examine DDN release and transfer at an even higher light intensity, e.g., 1100 and 1800 µE m^-2 s^-1 (approximately equal to full noon sunlight on a cloudless day) (Lu et al., 2018), at which the stable exponential growth of T. erythraeum and Synechococcus could not be achieved due to a strong light inhibition effect, we conducted short-term (24-h) mixed incubations that were enriched with ¹⁵N.

In the monoculture and co-cultures, T. erythraeum growth was monitored by daily measurements of the Chl a concentration. Briefly, T. erythraeum cells were collected by filtration onto 3-μm pore size polycarbonate membrane filters (PC, Millipore, Burlington, MA, USA). In the co-culture systems, because the cell size of Synechococcus was typically less than 1.5 μm, Synechococcus could not be trapped on the 3-μm PC filters. The filters were heated at 65°C for 6 min in 90% (vol/vol) methanol. After extraction, the filters were removed, and cell debris was pelleted by centrifugation. The Chl a concentration was subsequently determined by a spectrophotometric analysis following the method described by Demarsac and Houmard (1988). To count T. erythraeum cell numbers, photographs of T. erythraeum were taken using a camera (DS126281, Canon, Tokyo, Japan) connected to an inverted microscope (CKX41, Olympus, Tokyo, Japan). The total length of filaments in 1 mL culture were measured, and the cell number of ~20 filaments was counted. The average length of cells was obtained by dividing the total length of the measured filaments by their total cell number. The cell density of the culture was then calculated by dividing the total length of filaments in 1 mL culture by the average cell length. The Chl a per cell was calculated by dividing the Chl a concentration by the cell density.

The growth of C. watsonii and Synechococcus was monitored daily by measuring cell abundance using flow cytometry (Accuri™ C6, BD Biosciences, Franklin Lakes, NJ, USA). Briefly, samples were collected in 2 mL centrifuge tubes and preserved in freshly prepared 0.2-μm-filtered glutaraldehyde (0.5% vol/vol final concentration). After fixation in the dark for 15 mins, samples were frozen in liquid nitrogen and stored at -80°C until analysis. Synechococcus and C. watsonii cells were distinguished and quantified based on forward scatter and red fluorescence (670 nm).

Specific growth rates were calculated from the linear regressions of the natural logarithm of Chl a concentrations or cell densities versus time during the exponential growth phase. Each growth curve included four data points to ensure accuracy and reliability in the linear regressions.

Particulate N₂ fixation was measured using the ¹⁵N gas dissolution method (Mohr et al., 2010), while concurrently, a C fixation assay was conducted utilizing NaH¹³CO₃ (99 atom% ¹³C, Cambridge Isotope Laboratories, Cambridge, UK). The ¹⁵N₂ gas (98.9 atom%, Cambridge Isotope Laboratories, Lot #: I-21065/AR0664758) was tested and confirmed to be non-contaminated following the method of Yu et al. (2024). To prepare the ¹⁵N-enriched water, 5 mL of ¹⁵N₂ gas was dissolved into 500 mL of degassed seawater. Incubations were conducted in duplicate in acid-cleaned 1-L Nalgene polycarbonate bottles. Each bottle was spiked with 30 mL of ¹⁵N₂-enriched water and NaH¹³CO₃ solution to a final concentration of 200 μM, followed by incubation in an algae chamber for 24 h. The final ¹⁵N enrichment [100×¹⁵N/(¹⁵N+¹⁴N), atom%] of the N₂ pool in the incubation bottles was measured using a Membrane Inlet Mass Spectrometer (MIMS), yielding a value of 1.29 ± 0.11 atom% (n = 11). Then, cells were filtered onto 25 mm pre-combusted (450°C for 4 h) GF/75 filters (Advantec, Eden Prairie, MN, USA). Cells not enriched with ¹⁵N and ¹³C were also collected to establish the baseline enrichments for biomass ¹⁵N and ¹³C. All filters were acid fumed, dried, and then analyzed using an EA IsoLink™ IRMS system (Flash IRMS elemental analyzer coupled to a Delta V isotope ratio mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA).

The N and C contents of the GF/75 filter blanks were 0.06 ± 0.00 μmol and 1.16 ± 0.06 μmol, respectively, consistently lower than the N (>1 µmol) and C (>10 µmol) contents of the measured samples. The natural ¹⁵N and ¹³C enrichments of co-culture samples were ~0.366 atom% and ~1.081 atom%, respectively, which are significantly lower than the values in the samples spiked with ¹⁵N₂ and NaH¹³CO₃ (¹⁵N>0.654 atom%, ¹³C>2.724 atom%). The average reproducibility of the ¹⁵N and ¹³C measurement of the USGS-40 standard was ± 0.0002 atom% and ± 0.0005 atom% (n = 18), respectively. The ¹⁵N and ¹³C fixation rates were calculated based on methods described by Montoya et al. (1996) and Hama et al. (1983), respectively.

The total N₂ fixation rates were the total formation rates of DDN, including both the particulate and dissolved fractions.

To determine the DDN released to the dissolved fractions, 50 ml of the incubation waters, both with and without ¹⁵N enrichment, were filtered through 0.22 µm pore size Millex-GP syringe filters (Millipore Express PLUS membrane, Millipore). The filtrates were preserved at -20°C for subsequent measurement of the concentration and ¹⁵N enrichment of total dissolved nitrogen (TDN).

For measurement of TDN concentration, TDN was oxidized to NO₃ ^- using a purified persulfate oxidizing reagent (POR, ACS-grade, Merck, Rathway, NJ, USA) in a 12 mL 450°C pre-combusted borosilicate glass tube (Knapp et al., 2005). The POR was recrystallized four times and prepared as alkaline POR by dissolving 6 g K₂S₂O₈ and 6 g NaOH (ACS-grade, Merck) in deionized water to a final volume of 100 mL. The residual NO₃ ^- concentration in the POR (POR blank) was determined to be less than 2 μmol L^-1. Following oxidation, the sample pH was adjusted to 7–8 using 6 N HCl. The concentrations of the resulting NO₃ ^- were measured by a chemiluminescent analysis, with a detection limit of 0.5 μmol L^-1 (Braman and Hendrix, 1989).

Isotopic analyses of ¹⁵N enrichment of TDN were conducted using the denitrifier method, which involves an isotopic analysis of the nitrous oxide produced by denitrifying Pseudomonas aureofaciens (Sigman et al., 2001). These analyses were performed on a Gasbench-Isotopic Ratio Mass Spectrometer (Delta V, Thermo Fisher Scientific). The rate of DDN released into the dissolved pool was calculated following Bonnet et al. (2016c):

where ¹⁵N_ex is the ¹⁵N enrichment of the TDN fraction after 24 h of incubation relative to the time zero value, TDN_con is the measured TDN concentration, and ¹⁵N_sr is the ¹⁵N enrichment of the source N₂ pool in the incubation bottles (as mentioned above).

For flow cytometry sorting (to separate the Synechococcus from the co-cultured diazotrophs), 0.3 to 0.7 L of co-culture algae were concentrated onto 0.22 µm pore size polycarbonate filters (47 mm) using a Nalgene polysulfone filtration unit (Item#: 300−4050, Thermo Fisher Scientific). Both the filtration unit and membrane were acid-cleaned prior to use. The cells on the filter were then resuspended into 4.5 mL of filtered seawater in a cryovial, where they were then fixed and preserved using glutaraldehyde (final concentration of 0.5% vol/vol) that had been filtered through a 0.2 μm filter. After fixation in the dark for 15 min, samples were frozen in liquid nitrogen and stored at -80°C until analysis.

Cell sorting was conducted following the method described by Baer et al. (2017), using a BD FACSAria™ III flow cytometer equipped with 488 and 561 nm lasers and detectors for forward and side scatter at 692 and 530 nm, respectively. Synechococcus populations were determined based on forward scatter and orange fluorescence (530 nm). Pre-filtered 30‰ NaCl (CAS: 7647−14−5, pure-grade, Sigma-Aldrich, St. Louis, MO, USA) solution was used as sheath fluid. The sorted populations were collected in 15 mL high-clarity polypropylene conical tubes (FALCON, Corning, NY, USA) and subsequently filtered onto pre-combusted GF/75 filters. Samples were then frozen in liquid nitrogen and stored at -80°C for further analysis.

The PON and ¹⁵N of the sorted cells were analyzed using the persulfate oxidation method coupled with the denitrifier method, respectively, as described above. The rate of DDN transferred to Synechococcus was calculated as follows:

where ¹⁵N_syn is the ¹⁵N enrichment of Synechococcus after 24 h of incubation relative to the time zero value, PON_con is the particulate organic nitrogen content of the sorted Synechococcus (fmol cell^-1), A is the abundance of Synechococcus, and ¹⁵N_sr is the ¹⁵N enrichment of the source N₂ pool in the incubation bottles.

Filter and sheath blanks were measured ( Supplementary Table S2 ) and subtracted from each analysis of mass. Based on the filter and sheath blank results, as well as the Synechococcus cellular N quota, a minimum of 2.5 × 10⁷ Synechococcus cells were sorted to meet the quantifiable requirement.

The R software (version 4.3.0) was used to analyze data and establish the significance of differences based on a Welch two sample t-test or one-way ANOVA in combination with a Tukey post hoc test. A significance level of p < 0.05 was applied.

The T. erythraeum and C. watsonii monocultures were grown with similar growth rates of ~0.5 d⁻¹ under a light intensity of 200 μE m⁻² s⁻¹ ( Table 2 ). The total N₂ fixation rate (including fixed N in the particulate and dissolved phases) of T. erythraeum was either normalized to cell number (cell specific N₂ fixation rate) or C biomass (C-specific N₂-fixation rate), and was significantly higher than that of C. watsonii (t-test, p < 0.05, Table 2 ). Additionally, T. erythraeum had a significantly lower C:N and C-fix: N₂-fix ratios compared to C. watsonii (t-test, p < 0.01, Table 2 ), suggesting that T. erythraeum was less efficient than C. watsonii in using the fixed N to support its own C fixation.

The monoculture of Synechococcus sp. grew exponentially with a rate of 0.91 d⁻¹ in the medium fortified with sufficient nitrate (100 μM) and under a light intensity of 200 μE m⁻² s⁻¹ ( Supplementary Figure S1 ). Prior to inoculation into the exponentially growing T. erythraeum or C. watsonii cultures, Synechococcus was acclimated to an inorganic N-free medium for 2 days until cell growth ceased ( Supplementary Figure S1 ).

To establish co-culture systems in which diazotrophs and Synechococcus all sustained exponential growth, the inoculation ratios were optimized in the preliminary experiments. It was found that at inoculation ratios of Synechococcus: T. erythraeum = ~6:1 (cell number: cell number), Synechococcus and T. erythraeum grew exponentially at growth rates of 0.33 ± 0.03 and 0.43 ± 0.02 d^-1, respectively, under a light intensity of 200 μE m⁻² s⁻¹ ( Figure 1 ). Therefore, the T. erythraeum fixed N supported the growth of Synechococcus and the growth of Synechococcus slightly inhibited the growth of T. erythraeum by about 20% (0.43 d^-1 in co-culture vs 0.53 d^-1 in monoculture). Additionally, the growth rate of Synechococcus in this co-culture system was one third of the maximum growth rate under N-replete conditions and the same light intensity ( Supplementary Figure S1 ), suggesting a N-limitation of Synechococcus in the co-culture system. For the C. watsonii−Synechococcus co-culture system, the inoculation ratio of Synechococcus: C. watsonii was 0.003:1 (cell number: cell number), for both strains to sustain exponential growth at growth rates similar to those in the T. erythraeum-Synechococcus co-culture system ( Figure 1 ). The huge difference in the inoculation ratios in the two co-culture systems was mainly due to the differences in the cell specific N₂ fixation rates of the two diazotrophs, i.e., 0.31 pmol N cell^-1 d^-1 for T. erythraeum and 0.02 pmol N cell^-1 d^-1 for C. watsonii, respectively ( Table 1 ). Additionally, it is likely that the two diazotrophs released different amounts and speciation of DDN to support the growth of non-diazotrophic phytoplankton.

On the 3^rd day after initiating the T. erythraeum−Synechococcus and C. watsonii−Synechococcus co-culture systems (3^rd day in Figure 1 ), ¹⁵N enriched seawater was added and the culture bottles were continually incubated under the same growing conditions for 24 h. The DDN released to the dissolved phase and DDN transferred to Synechococcus were measured. In the two co-culture systems, the diazotrophs reached a similar biomass on the 3^rd day, i.e., 516 ± 12 and 619 ± 27 μmol C L^-1 for T. erythraeum and C. watsonii, respectively. The ¹⁵N in diazotrophic (0.902 atom% and 0.738 atom% for T. erythraeum and C. watsonii respectively) and Synechococcus biomass (> 0.472 atom%), as well as in the dissolved pool (> 0.386 atom%) after 24 h of incubation were significantly enriched compared with the abundance of the natural isotope (~0.366 atom%, t-test, p < 0.05, Supplementary Table S1 ). The total volumetric N₂ fixation rate of T. erythraeum (34.9 ± 2.7 µmol N L^-1 d^-1), including the particulate fraction collected on the filter and the fraction released into the dissolved pool, was nearly double that of C. watsonii (18.9 ± 3.2 µmol N L^-1 d^-1). The majority of the total DDN (>95%) was contained within the diazotrophs themselves.

The rate of DD¹⁵N release into the dissolved pool (calculated by Equation 1) in the T. erythraeum−Synechococcus co-culture system was 507 ± 101 nmol N L^-1 d^-1, with no significant difference compared to that of the C. watsonii−Synechococcus co-culture (489 ± 49 nmol N L^-1 d^-1, t-test, Figure 2A ). However, the percentage of the released DD¹⁵N relative to the total fixed N was slightly higher in C. watsonii (2.61%) compared to T. erythraeum (1.45%) (t-test, p < 0.01, Figure 2B ). The volumetric rates of T. erythraeum DD¹⁵N transferred to Synechococcus (calculated by Equation 2) were significantly higher than those of the C. watsonii DD¹⁵N (t-test, p < 0.01, Figure 2A ). Additionally, T. erythraeum had a significantly higher DD¹⁵N transfer efficiency (i.e., the proportion of the total fixed N, 0.36%) than C. watsonii (< 0.01%, t-test, p < 0.01, Figure 2B ) in the co-cultures. Whereas, the total DDN release fractions (DDN released to the dissolved pool plus the DDN transferred to Synechococcus) did not differ significantly between the two co-culture systems.

To explore the impact of light intensity on DDN release and transfer, we conducted additional co-cultures of T. erythraeum and Synechococcus under light intensities of 70 and 750 μE m^-2 s^-1, and compared the results to the treatment under the 200 μE m^-2 s^-1 condition ( Table 1 ). The initial inoculation ratios of T. erythraeum and Synechococcus were kept similar under the different light treatments (5−8:1, Synechococcus cell number: T. erythraeum cell number, Table 1 ). T. erythraeum and Synechococcus grew exponentially under the three light intensities. The growth rates and total N₂ fixation rates of T. erythraeum were significantly lower under 70 and 750 μE m^-2 s^-1 than under 200 μE m^-2 s^-1, suggesting that 70 and 750 μE m^-2 s^-1 were light-limited and light-inhibited conditions, respectively, for the growth and N₂ fixation of T. erythraeum ( Table 1 ). The growth rates of Synechococcus were lower under the low light conditions of 70 μE m^-2 s^-1 than 200 μE m^-2 s^-1, with consistently lower total N₂ fixation rates of T. erythraeum under low light conditions. In contrast, the growth rates of Synechococcus increased when light intensity increased to 750 μE m^-2 s^-1, under which N₂ fixation of T. erythraeum was inhibited by the high light conditions ( Table 1 ). In line with these results, T. erythraeum exhibited a higher DD¹⁵N transfer efficiency under 750 μE m^-2 s^-1 than under 70 and 200 μE m^-2 s^-1 ( Figure 3 ).

Consistently, the highest percentage of DDN released into the dissolved phase was found under a light intensity of 750 μE m^-2 s^-1 (3.19%), although no statistically significant difference was found among the three light intensities (one-way ANOVA, p > 0.05, Figure 3 ). As a result, the total released fraction (release to the dissolved phase plus transfer to Synechococcus) was highest under 750 μE m^-2 s^-1 ( Figure 3 ). To confirm that a high light intensity increased the DDN release, we conducted short-term (24-h) co-culture incubations under light intensities of 1100 and 1800 μE m^-2 s^-1, that were approximately equivalent to full noon sunlight on a cloudless day (Lu et al., 2018). Under these two high light conditions, T. erythraeum and Synechococcus could not grow exponentially due to strong light inhibition effects; therefore, only short-term incubations were conducted. The results showed that although the total N₂ fixation rates were very low under the high light conditions of 1100 and 1800 μE m^-2 s^-1, the portion of fixed N released to the dissolved pool increased with increasing light intensity and reached ~25% under 1800 μE m^-2 s^-1 ( Figure 4 ).

In both the monoculture and co-cultures, T. erythraeum exhibited higher cell-specific and C-specific N₂ fixation rates than C. watsonii under the same culture conditions ( Tables 1 ; 2 ). These findings were aligned with previous reports in which N₂ fixation rates were measured using the conventional acetylene reduction method (Knapp et al., 2012) and the novel nanoSIMS analysis (Berthelot et al., 2016). However, T. erythraeum was less efficient at using the fixed N to support its own C fixation than C. watsonii, as indicated by the lower C-fix:N₂-fix ratios compared to C. watsonii ( Table 2 ). As filamentous cyanobacterial diazotrophs, only 15−20% of its cells within a Trichodesmium trichome are diazocytes that are capable of N₂ fixing. It was proposed that the new N fixed by diazocytes would be actively released into the surrounding environment and subsequently taken up by the vegetative cells in the rest of the filaments or by other phytoplankton (Berman-Frank et al., 2003; Mulholland et al., 2004). However, C. watsonii are unicellular N₂ fixing cyanobacteria. Therefore, it is likely that T. erythraeum may release more of its fixed DDN to the surrounding environment and be less efficient in using its own fixed DDN.

After measuring DDN release, we did not find more DDN present in the dissolved phase in the T. erythraeum−Synechococcus co-culture compared to the C. watsonii−Synechococcus co-culture. The rate of DDN release was similar between the two species ( Figure 2 ), even though the volumetric N₂ fixation rate of T. erythraeum was twice as high as that of C. watsonii. Even when accounting for the DDN transferred to Synechococcus, the total amount of DDN excreted by T. erythraeum was similar to that of C. watsonii ( Figure 2 ). However, the DDN excreted by T. erythraeum was more efficient in supporting Synechococcus cell growth in the co-cultures ( Figure 2 ; Table 1 ). We propose that fixed N derived from T. erythraeum was likely more bioavailable for Synechococcus than that from C. watsonii. This was supported by the significantly higher DDN transfer efficiency of T. erythraeum (0.36%) compared to C. watsonii (< 0.01%) ( Figure 2 ). It was also reported that in artificially induced blooms in the western South Pacific, a T. erythraeum bloom resulted in a significantly higher ¹⁵NH₄ ⁺ enrichment in the dissolved pool than a C. watsonii bloom (Berthelot et al., 2016), suggesting a higher bioavailability of the fixed N released from T. erythraeum. Therefore, DDN released by T. erythraeum can contribute significantly to primary production by supporting the growth of non-diazotrophic phytoplankton (Campbell et al., 2005).

For the smaller unicellular C. watsonii, although the active release of its DDN played a minor role in supporting the growth of non-diazotrophic phytoplankton, it is more easily grazed by zooplankton than T. erythraeum and resulted in a more passive release of DDN (Caffin et al., 2018). Nevertheless, our co-culture systems provided a useful approach to compare the active DDN release and transfer by the two different diazotrophs. It should be noted that our observations of DDN release and transfer were the results obtained for 24-h of incubation after ¹⁵N enrichment. The turnover time of DON could be longer than 24-h (Bronk et al., 2007), therefore, the efficiency of DDN transfer may have been underestimated in our study systems.

Our study demonstrated the significant role of light intensity in controlling the release and transfer of DDN fixed by T. erythraeum. It was shown that T. erythraeum supported higher Synechococcus growth under a high light intensity of 750 μE m⁻² s⁻¹ ( Table 1 ), which aligned well with the increased percentage of total DDN released to the dissolved pool and transferred to Synechococcus ( Figure 3 ). Additionally, although even higher light intensities, e.g., 1100 and 1800 μE m⁻² s⁻¹, inhibited the growth and N₂ fixation of T. erythraeum, they further increased the portion of fixed N released to the dissolved pool ( Figure 4 ). It has been proposed that that the exudation of DDN in the form of NH₄ ⁺ and DON could serve as a potential electron sink to protect cells from photo-damage (Wannicke et al., 2009).

However, previous field incubation experiments have found that the fraction of DDN in the dissolved phase increased when the light intensity decreased to a very low level, e.g., 15 μE m⁻² s⁻¹ (Lu et al., 2018). We found no significant difference in the fraction of DDN in the dissolved phase among the three light treatments of 70, 200, and 750 μE m⁻² s⁻¹. The DDN in the dissolved phase was the net result of diazotroph DDN excretion and uptake by phytoplankton. Light intensity not only affects the DDN excretion rate, but also the phytoplankton N uptake rate (Wannicke et al., 2009; Han et al., 2023). Under a very low light intensity, such as 15 μE m⁻² s⁻¹, although diazotrophs could decrease DDN excretion, phytoplankton may be short of energy for N uptake, leading to an increase in percentage of DDN in the dissolved fractions (Lu et al., 2018; Han et al., 2023). These studies highlight the important role of light intensity in controlling the diazotrophic N₂ fixation rate and DDN release rate, as well as phytoplankton N uptake rates. Our results using the simple and well-controlled co-culture system clearly showed how light intensity affects T. erythraeum N₂ fixation, DDN release, and transfer.

Factors other than light can also affect diazotrophic DDN release. For example, iron and phosphorus limitation can affect the DDN release of Azotobacter vinelandii, T. erythraeum, or Nodularia (McRose et al., 2019; Wannicke et al., 2009; Schoffelen et al., 2019). Moreover, top-down controls such as viral lysis and grazing could stimulate passive DDN release and promote C export (Bonnet et al., 2016a; Kuznecova et al., 2020). Future studies should investigate the effects of these potential factors to obtain a comprehensive understanding of the fate and role of DDN in supporting marine primary production.