The diazotrophic marine cyanobacterium Trichodesmium is ubiquitous in tropical and subtropical seas, where it fixes both CO₂ and N₂, making it among the most biogeochemically significant microorganisms in the ocean (Moisander et al., 2010; Zehr, 2011; Walworth et al., 2018). It has been estimated that Trichodesmium may release approximately 30 to 50% of its newly fixed nitrogen (N), providing an important source of bioavailable N to other non-diazotrophic phytoplankton species in the N-limited subtropical marine ecosystems (Glibert and Bronk, 1994; Walworth et al., 2018). The global N input via N₂ fixation by Trichodesmium is estimated around 60–80 Tg N annually (Capone et al., 1997; Mahaffey, 2005; Carpenter and Capone, 2008), which contributes substantially to the annual global marine N₂ fixation estimated to be around 223 ± 30 Tg N (Shao et al., 2023).

Although Trichodesmium is not the sole diazotrophic cyanobacterium in the open ocean (Sohm et al., 2011; Gradoville et al., 2020; Hutchins and Capone, 2022), it is the most intensively explored cyanobacterium. Since it has been in culture from the early 1990s (Prufert-Bebout et al., 1993), numerous studies have focused on the eco-physiological responses of Trichodesmium to different environmental factors. Despite many studies emphasizing the physiology and biogeographical distribution of Trichodesmium, fewer have examined the fate of the N₂ fixed by Trichodesmium (diazotroph-derived N, DDN) in the ocean (Capone et al., 1997). Some of these studies have reported that Trichodesmium blooms may encourage the growth of other non-N-fixing organisms (Padmakumar et al., 2010; Basu et al., 2011). For instance, the abundance of bacteria and the growth of diatoms and dinoflagellates were found to be significantly increased during Trichodesmium blooms in the Eastern Arabian Sea (Padmakumar et al., 2010; Basu et al., 2011). Both field and laboratory data demonstrated that the co-occurrence of Trichodesmium spp. and diatoms might be driven by the transfer of N fixed by Trichodesmium spp. in the N-limited oligotrophic ocean (Chen et al., 2011). Similarly, a previous study showed that a unicellular dinoflagellate, Karenia brevis, could grow well in the culture medium supplemented with Trichodesmium exudates as the sole source of nitrogen (Mulholland, 2007).

In general, Trichodesmium helps sustain marine life via both the active release of key nutrients such as carbon and nitrogen, and passive release processes like cell death, virus-induced lysis, and grazing by heterotrophs. Hence, it plays a crucial role in the biogeochemical cycling of bioactive elements in contemporary oceans (Capone and Carpenter, 1982; Capone et al., 1997). However, it is unknown how the specific forms of nitrogen are transferred by Trichodesmium to non-N-fixing organisms. Moreover, there is a lack of quantitative information regarding the concentrations, composition, and fate of DDN. With the development of techniques such as nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with ¹⁵N₂ isotopic labelling and flow cytometry cell sorting, researchers have observed the transfer of DDN to the dissolved N pool and to non-diazotrophic plankton such as diatoms, dinoflagellates, and bacteria (Mulholland et al., 2004; Dore et al., 2008; Lenes and Heil, 2010; Chen et al., 2011). Previous reports have shown that the DDN transfer efficiency of Trichodesmium is higher compared to other diazotrophic cyanobacteria, indicating that Trichodesmium blooms are more efficient in promoting non-diazotrophic production in N-depleted areas (Konno et al., 2010; Benavides et al., 2013; Berthelot et al., 2016; Bonnet et al., 2016). However, this method based on isotopic conversion does not allow for accurate discrimination of nitrogen sources, as well as the assessment of the nitrogen utilization efficiency of non-nitrogen-fixing marine phytoplankton.

In addition to nitrogen-fixing cyanobacteria, many non-nitrogen-fixing cyanobacteria exist in nitrogen-deficient oceans, such as Synechococcus and Prochlorococcus, which are among the most abundant phytoplankton in the ocean (Flombaum et al., 2013). However, it is unclear whether a substantial fraction of nitrogen required for phytoplankton growth comes from DDN released by Trichodesmium. Previous research has concluded that the transfer of DDN to nearby plankton is mediated through the dissolved N pool, as diazotrophs release a significant fraction of their fixed N (10–50%) in the form of ammonium and dissolved organic nitrogen (DON) (Glibert and Bronk, 1994; Mulholland and Bernhardt, 2005; Konno et al., 2010; Benavides et al., 2013; Berthelot et al., 2015; Klawonn et al., 2020; Benavides et al., 2022). These studies suggest that although more than 50% of recently fixed N₂ is released by diazotrophs as DON, NH₄ ⁺ is the main pathway of DDN transfer from diazotrophs to non-diazotrophs. However, NH₄ ⁺ may not accumulate in the surrounding environment as it could be utilized by the diazotrophs themselves and the surrounding organisms as soon as it is produced (Klawonn et al., 2020). Instead, DON concentrations of 5 to 10 μM are common, making it the most abundant form of dissolved nitrogen (Mulholland et al., 2004; Benavides et al., 2013). This suggests that organic compounds exuded by diazotrophs can fuel primary production, and may have considerable effect on the composition of the plankton community in the oligotrophic ocean. The dominant non-nitrogen-fixing cyanobacteria in these ecosystems, including Synechococcus and Prochlorococcus, possess pathways to take advantage of DON sources, such as urea or dissolved free amino acids (Ludwig and Bryant, 2012; Veaudor et al., 2019; Muñoz-Marín et al., 2020). However, the underlying potential mechanisms have rarely been explored.

Trichodesmium often forms large macroscopic colonies and acts as relatively nutrient-rich substrate for a diverse microbial community through physical attachment and direct colonization in tropical and subtropical oligotrophic oceans (Eichner et al., 2023). A common feature of these associations is the passive transfer of fixed nitrogen (N), such as ammonia, urea, and other DON, from Trichodesmium to epibionts (Mulholland et al., 2004). On the other hand, epibionts assist the host Trichodesmium in iron and phosphorus acquisition, vitamin B12 exchange, small carbon compound catabolism, and detoxification of reactive oxygen species (Lee et al., 2017). Thus, it is difficult to maintain Trichodesmium in culture and unsuccessful to establish stable axenic cultures, due to its obligate dependency on associated organisms. In this study, Trichodesmium erythraeum IMS 101 has been maintained in the laboratory for decades and shown to harbor microbial communities similar to natural microbial populations (Lee et al., 2017). Previous meta-transcriptomics research has detected the community transcripts involved in the reduction of nitrate, nitrite, and nitrous oxide, and suggested that the associated organisms may play an important role in colony-level nitrogen cycling (Lee et al., 2018). To understand the significance of fixed nitrogen released by Trichodesmium to other non-diazotrophic microorganisms, it is prudent to consider the entire Trichodesmium-epibiont assemblage for the stable cohabitation of a diverse community within Trichodesmium consortia.

Although previous studies have shown that Trichodesmium can input nascent nitrogen sources into the habitat, there still lacks direct experimental evidence on whether Trichodesmium can drive phytoplankton growth (Glibert and Bronk, 1994; Mulholland et al., 2004). To investigate under what conditions and in what form Trichodesmium provides nitrogen sources for other cyanobacteria, we studied the growth and nitrogen-fixing gene expression of Trichodesmium under different nitrogen sources as well as nitrogen concentration conditions. The types and amounts of nitrogen sources secreted by the Trichodesmium symbiotic system under nitrogen-deficient conditions were analyzed, and marine Synechococcus sp. strain PCC 7002 was chosen to study the specific forms of nitrogen sources provided by Trichodesmium to other phytoplankton. We also validated this result using a mutant strain that lacked some of the nitrogen source utilization functions. Our results provide experimental evidence that the nitrogen-fixing cyanobacterium Trichodesmium can provide a certain amount of urea to the different species of marine phytoplankton around it, a nitrogen source that has been previously overlooked.

Trichodesmium may fix dinitrogen (N₂) in the oligotrophic near-surface regions of ocean, alleviating N limitation and encouraging a more diverse plankton community. The nitrogen fixed by Trichodesmium has been predicted to provide up to 50% of the new nitrogen-supporting open-ocean food webs in the subtropical North Pacific Gyre (Dore et al., 2002). Many reports have demonstrated that Trichodesmium fixes nitrogen under nitrogen deficient conditions and high concentrations of exogenous inorganic nitrogen in the environment may significantly decrease the N₂ fixation rate of Trichodesmium (Holl and Montoya, 2005; Sandh et al., 2011; Eichner et al., 2014; Walworth et al., 2018). To date, most diazotrophic nitrogen assimilation studies have focused on the relationship between N₂ fixation and uptake of nitrate, ammonia, and amino acid (Ohki et al., 1991; Mulholland et al., 1999, Mulholland et al., 2001). The absorption and utilization characteristics of Trichodesmium for various nitrogen compounds are largely unknown. In this study, the growth and expression of nifH gene (encodes nitrogen-fixing enzymes of Trichodesmium) were analyzed in the presence of different types of nitrogen sources in varying concentrations. The results demonstrated that urea (common DON in the water) supply at a certain concentration had little effect on nifH gene expression in Trichodesmium, as compared with the similar concentration of NH₄ ⁺ and NO₃ ^- (mainly dissolved inorganic nitrogen (DIN) in the water) supply. Moreover, urea supply even could promote the growth of Trichodesmium. Similarly, recent research has demonstrated that carbon-, nitrogen- and phosphorus-rich dissolved organic matters (DOMs) enhance N₂ fixation rates and nifH gene expression in natural Trichodesmium colonies (Stolte et al., 2006; Letscher and Moore, 2015; Benavides et al., 2018). It was speculated that in the presence of exogenous DON, Trichodesmium could continue to fix nitrogen and release new nitrogen sources to improve primary productivity in marine ecosystems.

In this study, the results demonstrated that DON accounted for the majority, while NH₄ ⁺ contributed to only a small proportion of Trichodesmium derived N left in the filtrate in the late exponential growth phase. This result was consistent with previous studies reporting low DIN and high DON in the culture filtrate of Trichodesmium (Mulholland et al., 2001; Glibert et al., 2008). It must be noticed that whether in the lab culture or the field, we should consider the entire Trichodesmium-epibiont assemblage as a whole for the stable cohabitation of a diverse community within Trichodesmium consortia. Thus, the main form of nitrogen source present in the filtrate was DON after equilibrium between nitrogen fixation by Trichodesmium and consumption of nitrogen source by the Trichodesmium consortia. However, compared to the released DON, the remaining NH₄ ⁺ concentration in the surrounding environment of Trichodesmium was extremely low (Berthelot et al., 2016). This phenomenon may be explained by the rapid release and uptake of NH₄ ⁺ by the Trichodesmium community as a potential major recycling intermediate of Trichodesmium-fixed N₂ in the culture system, which facilitates the rapid turnover of the nitrogen pool and leaves little residue in the culture medium (Mulholland et al., 1999, 2001). Whereas DON was the main nitrogen resource accumulated in the medium, contributing a large fraction of the N pool that could be utilized by other microorganisms in the surrounding environment.

Few studies evaluated the bioavailable proportion of Trichodesmium-derived DON and the compounds within the uncharacterized DON pool that were utilized by other phytoplankton (Mulholland et al., 2004). DON has been largely understood by extrapolating the data obtained from the studies of dissolved organic carbon flow or through studies of individual compounds, such as urea or dissolved free amino acids (Glibert and Bronk, 1994; Mulholland et al., 2004; Finzi-Hart et al., 2009). In the present study, the nitrogen forms released by Trichodesmium were evaluated in the culture medium without exogenous N and the proportion of urea in the total nitrogen was found to be more than 20%. These results indicate that urea may play an important role in marine nitrogen cycle and serve as nitrogen resource for transferring nitrogen to auto- and heterotrophic plankton communities in the surrounding environment.

Many non-nitrogen-fixing cyanobacteria, such as Synechococcus and Prochlorococcus, co-exist with nitrogen-fixing cyanobacteria in nitrogen-deficient oceans (Flombaum et al., 2013; Masuda et al., 2022). Synechococcus 7002, originally isolated from the mud of the Atlantic coast, can be genetically manipulated and grown in axenic cultures. Recent studies have shown that the distribution of Synechococcus in coastal regions is far more extensive than previously thought (Lee et al., 2017; Yong et al., 2023). By using the macro-genome database of Tara Ocean, the presence of Synechococcus 7002 has been detected in the sights where Trichodesmium exists. This shows that Trichodesmium and Synechococcus 7002 (at least strains similar to Synechococcus 7002 due to the inaccuracy of bioinformatics prediction) possibly have a close association in the oceans ( Figure 1 ; Supplementary Figure S3 ). To further reveal the potential interaction between Trichodesmium and the “consumer” of its secreted nitrogen, Synechococcus 7002 was co-cultured with Trichodesmium and its secreted filtrate was used as the sole nitrogen resource. When Trichodesmium reached a dynamic equilibrium with Synechococcus cells in the culture system, both in terms of biomass and nitrogen turnover, chronically nitrogen-deficient Synechococcus 7002 recovered from chlorosis to green cells ( Figure 1E ). This implied that nitrogen fixation by Trichodesmium provided an important source of nitrogen for the recovery of Synechococcus 7002. In mono-cultures, both Synechococcus 7002 and Trichodesmium preferred urea as a nitrogen source than other inorganic nitrogen sources (e.g., ammonium and nitrate). Inhibition of Trichodesmium growth was observed at high dose of exogenous inorganic nitrogen (NO₃ ^-, NH₄ ⁺), but the growth increased at a higher concentration of urea. Synechococcus 7002 showed good recovery after a prolonged period of nitrogen deficiency, when exogenous nitrogen sources were supplied. Among the added exogenous nitrogen sources, ammonium and urea were preferred by Synechococcus 7002, which was consistent with the previous study reporting ammonia as the preferred microbial nitrogen source for most, if not all, cyanobacteria (Walworth et al., 2018). After the addition of Trichodesmium IMS 101 filtrate to Synechococcus 7002 culture, the biomass of nitrogen-deficient dormant Synechococcus 7002 also increased significantly ( Figure 6 ). These results suggest that the nitrogen source released by Trichodesmium can be directly utilized by Synechococcus 7002 for its growth.

Some studies investigated the transfer of diazotroph-derived nitrogen to non-diazotrophic planktonic communities (Chen et al., 2011; Sipler et al., 2013). For instance, Chen et al. (2011) conducted ¹⁵N tracing experiments and revealed the transfer of newly fixed N from Trichodesmium to diatoms. Sipler et al. (2013) used the FT-ICR MS method to detect the changes in DON concentration and the composition of N compound showed that Trichodesmium sp. provided a sufficient source of nitrogen to directly or indirectly support Karenia brevis blooms. However, the potential mechanism involved in the transfer of Trichodesmium-derived DON such as urea and amino acids to non-diazotrophic plankton need to be further studied at physiological and molecular level. In the present study, the biomass of Mut-ureA strain was around 20% lower than the biomass of the wild type Synechococcus. This result indicated that urea provided a fraction of nitrogen source that Synechococcus could obtain from Trichodesmium filtrate. The physiological study of genetic mutants provided direct evidence that chronically nitrogen-starved marine Synechococcus was able to access urea (produced during nitrogen fixation by Trichodesmium) as the nitrogen source, although other forms of nitrogen released by Trichodesmium also had positive effects on the growth of Synechococcus, which account for ~80% of the biomass accumulation. A previous study examined the transcriptional profile of cyanobacteria co-existing in Trichodesmium populations, which was primarily dominated by Synechococcus (Lee et al., 2018). The study reported that the cyanobacteria allocated more of their transcriptional pool to ammonium (amtB) and urea (urtA) transport, with protein products also detected during urea transport, indicating the potential utilization of Trichodesmium-derived urea by Synechococcus (Lee et al., 2018). In the present study, wild-type and Mut-ureA strains of Synechococcus 7002 were used in this study to understand the relationship between Trichodesmium and non-nitrogen-fixing Synechococcus linked by the fixed and released nitrogen of Trichodesmium. The results showed that the Trichodesmium-derived urea contributed at least part of the nitrogen source for the growth of phytoplankton populations. This suggests that urea exuded by diazotrophs can fuel the primary production and may have a considerable impact on the composition of the plankton community in the oligo-trophic oceans.

Long-term evolution has led to the development of different nitrogen uptake and utilization mechanisms as well as the nitrogen source preferences of various marine phytoplankton. This may challenge the potential of Trichodesmium as an important supplier of nitrogen sources to promote marine primary productivity (Moore et al., 2002; Zubkov et al., 2003; Esteves-Ferreira et al., 2018; Li et al., 2023). This study emphasized the crucial role of Trichodesmium-derived DON, especially urea, as new nitrogen to support the growth of phytoplankton in the surrounding environment, represents an important pathway of N and C export to the deeper parts of tropical and subtropical oceans. Further studies are needed to investigate the fate of other dominant Trichodesmium-derived N forms and to highlight the complex interactions of diazotrophs with their environment.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

S-QL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. YX: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. H-LH: Data curation, Investigation, Methodology, Validation, Visualization, Writing – review & editing. ZL: Formal analysis, Investigation, Methodology, Software, Writing – review & editing. T-RS: Formal analysis, Investigation, Software, Writing – review & editing. H-YG: Data curation, Investigation, Methodology, Software, Writing – review & editing. X-WW: Conceptualization, Funding acquisition, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. H-BJ: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.