Biological dinitrogen (N₂) fixation, the conversion of dissolved N₂ into ammonia, can represent a critical import of reactive nitrogen to the pelagic environment (Karl et al., 2002). This process is carried out by specialized prokaryotic microorganisms termed diazotrophs (Zehr and Turner, 2001). Aquatic studies have traditionally focused on photoautotrophic cyanobacterial diazotrophs inhabiting oligotrophic and sunlit environments where energy is made available via photosynthetic carbon fixation (Zehr, 2011). However, during the last decade it has become evident that non-cyanobacterial diazotrophs (NCDs; see Box 1) have an almost ubiquitous distribution in pelagic environments (Farnelid et al., 2011; Langlois et al., 2015; Geisler et al., 2020; Hallstrøm et al., 2021; Messer et al., 2021). For instance, the presence and/or expression of the nitrogenase gene (nifH) by NCDs has been reported from low latitude open oceans (Halm et al., 2011; Moisander et al., 2014; Langlois et al., 2015) to environments previously not regarded as suitable for N₂ fixation such as eutrophic rivers, estuaries and coastal waters (Mulholland et al., 2012; Bentzon-Tilia et al., 2015; Geisler et al., 2020; Hallstrøm et al., 2021), the aphotic deep sea (Rahav et al., 2013; Benavides et al., 2015), and nutrient-rich arctic waters (Harding et al., 2018). A recent study suggested that some nifH genes are not functional (Mise et al., 2021). Yet, these genes relate to obligate anaerobic bacteria and their prevalence in the marine pelagic environment is likely minor. The activity of NCDs has been indirectly inferred by experimental manipulations that inhibit photoautotrophic activity (Rahav et al., 2016; Benavides et al., 2018b; Geisler et al., 2019, 2020) and from environments putatively void of cyanobacteria such as aphotic waters (Hamersley et al., 2011; Rahav et al., 2013; Benavides et al., 2015). Still, these methods cannot measure NCD-specific N₂ fixation rates unambiguously. Thus, despite of the widespread distribution and activity of NCDs, their contribution to aquatic nitrogen cycling remains elusive (see reviews: Riemann et al., 2010; Bombar et al., 2016; Moisander et al., 2017; Benavides et al., 2018a; Marcarelli et al., 2022).

The marine water column is generally well oxygenated (except for oxygen minimum zones) and poor in labile organic matter (Arrieta et al., 2015; Santinelli, 2015), whereas the aphotic zone is rich in reactive nitrogen (e.g., Cavender-Bares et al., 2001). Therefore, the wide distribution of NCDs in these habitats with apparent unfavorable conditions for diazotrophy represents a lingering enigma. In this mini-review, we compile recent reports related to NCDs and focus on those associated with aggregates. We argue that the plot thickens [sensu (Azam, 1998)], and that compelling evidence supports the idea of planktonic aggregates as important microenvironments suitable for NCD N₂ fixation. We emphasize the need for direct in situ measurements of aggregate-associated, NCD-specific N₂ fixation, and provide guidelines for how these can be obtained in future studies. We note that this review paper focuses on marine and estuarine environments, as most data are available from such environments, but acknowledge that NCDs are also found in freshwater ecosystems (Coyne et al., 2020; Fernandez et al., 2020; Geisler et al., 2020).

Aggregates are ubiquitous throughout marine and freshwater environments (Alldredge and Gotschalk, 1988; Waite et al., 2000). They are formed by the coagulation of live and dead plankton material (Smith et al., 1992; Grossart and Ploug, 2000; Piontek et al., 2009; Daly et al., 2016). The elevated micronutrient and macronutrient concentration relative to the surrounding waters fosters colonization by dense communities of prokaryotes (del Giorgio and Cole 1998; Simon et al., 2002; Bar-Zeev and Rahav 2015), making aggregates ‘hot spots’ of intense microbial activity (Azam and Long, 2001). More than three decades ago, Hans Paerl and co-workers (Paerl, 1985; Paerl and Prufert, 1987) suggested that NCD N₂ fixation may take place in low oxygen microzones within aggregates. This idea was reiterated in several later studies (Riemann et al., 2010; Sohm et al., 2011; Bombar et al., 2016), but has been substantiated only most recently (see below).

In the past, and especially during the last decade, evidence has accumulated for the association of NCDs with aquatic organisms and aggregates. NCDs have been isolated from copepods (Proctor, 1997) and nifH genes have been amplified and sequenced from copepods and euphausiids (Braun et al., 1999; Scavotto et al., 2015), and dinoflagellates (Farnelid et al., 2010). Moreover, individual and bulk aggregates collected with sediment traps deployed at 150 m depth in the open ocean contained nifH gene sequences of diverse NCDs (Farnelid et al., 2018). The prevalence of NCDs on aggregates has also been reported using metagenomics sequencing. In the Tara oceans dataset, representing 197 globally distributed pelagic oceanic metagenomes, the putative heterotrophic Planctomyces and Proteobacteria accounted for ~25% of the nifH reads obtained from the 180 to 2,000 μm size-fraction (Karlusich et al., 2021). Moreover, metagenome assembled genomes representing NCDs occurred in the 5–2,000 μm planktonic size-fractions (Delmont et al., 2021). Finally, one of the most widely distributed NCDs, Gamma-A, showed a ubiquitous presence in nifH genes across the North Atlantic Ocean quantified by qPCR in the >3 μm fraction (Benavides et al., 2016). This Gamma-A was also found in metatranscriptomes from the 3 to 2,000 μm size-fraction in the Tara oceans dataset, suggesting a filamentous or aggregate-attached lifestyle for this putative heterotrophic bacterium (Cornejo-Castillo and Zehr, 2020). Hence, both PCR-dependent and -independent approaches suggest the presence and/or activity of NCDs on aggregates.

Experimental data also suggest aggregate-associated N₂ fixation by NCDs. In an early study from the Chesapeake Bay, United States, experiments by Guerinot and Colwell (1985) suggested that isolated strains of NCDs could fix N₂ in the presence of plankton and particulate matter. In an experiment with aggregates from the Southern Indian Ocean, nifH genes related to Deltaproteobacteria were enriched in metatranscriptomes from experimental incubations with aggregates relative to controls without aggregates (Debeljak et al., 2021). Similarly, N₂ fixation was stimulated in seawater from a Danish nutrient rich estuary and the Mediterranean Sea by amendment with natural aggregates (Pedersen et al., 2018) or a transparent exopolymer aggregate analog (gum-xanthan; Rahav et al., 2016), respectively. Hence, the presence of aggregates appears to stimulate N₂ fixation by NCDs. Finally, presence of NCDs was recently documented on aggregates by immunolabeling of the nitrogenase enzyme while at the same time superimposing the aggregate matrix, total bacteria and cyanobacteria (Figure 1A). Collectively, the above-mentioned findings suggest that NCDs benefit from colonizing aggregates. Yet, our mechanistic understanding of how aggregates support N₂ fixation by NCDs is still rather limited.

Theoretical considerations as well as experimental and field observations indicate that aggregates provide several conditions, which at least ephemerally, can support N₂ fixation by NCDs: (1) Low oxygen conditions: Nitrogenase, a central enzyme for N₂ fixation, is irreversibly damaged by molecular oxygen (Goldberg et al., 1987); however, aerobic respiration by bacteria that colonize the aggregate combined with slow diffusion rates (depending on the size and density of the particle) leads to local reduction in oxygen concentrations (Alldredge and Cohen, 1987; Paerl and Prufert, 1987; Ploug et al., 1997; Simon et al., 2002; Klawonn et al., 2015). The low oxygen levels in some parts of the aggregate vary from 50% to 90% air-saturation to anaerobic conditions on some occasions inside compact and large (few mm) aggregates (Ploug et al., 1997; Ploug, 2001; Klawonn et al., 2015). Consequently, low-oxygen microzones within aggregates likely provide loci where the nitrogenase enzyme is protected from oxygen (Figure 1B). (2) Metabolic energy: Aggregates are characterized by elevated levels of labile organic carbon relative to the surrounding waters and rapidly become colonized by diverse bacteria. Enzymatic hydrolysis of the aggregate matrix allows ample carbon and nutrient supply and extensive microbial growth (Ploug and Grossart, 2000). This organic-rich microenvironment can, therefore, support the high energy requirements associated with diazotrophy by NCDs. (3) Reactive nitrogen availability: The high C:N ratio of aggregates (Figure 1C), and the consequent reduction in nitrogen availability due to microbial growth, may provide NCDs a competitive edge over other bacteria unable to fix N₂. (4) Trace metal and phosphorus availability: Diazotrophy requires trace metals such as iron (Berman-Frank et al., 2001) and molybdenum (Marino et al., 2003). Since aggregates usually contain higher concentrations of trace metals than ambient water (Jackson and Burd, 1998; Engel et al., 2004), inhabiting diazotrophs may gain efficient access to these nutrients, in particular in the presence of increased microbial activity. Based on these observations, it may be surmised that aggregates can provide conditions that are beneficial for N₂ fixation by NCDs.

Aggregates may provide favorable conditions for NCDs under various conditions in marine and freshwater environments. Yet, how these conceivably ephemeral conditions develop on aggregates and how NCDs exploit them is currently unclear. Based on the overall emerging picture outlined above, and recent experimental (Martínez-Pérez et al., 2018; Paerl et al., 2018) and modeling work (Chakraborty et al., 2021), we suggest a conceptual framework for N₂ fixation by NCDs associated with aggregates (Figure 1D).

In the photic, well-oxidized zone, newly formed aggregates are sparsely colonized by microorganisms, thus limited respiration is expected. At that time, diffusion of oxygen from the surrounding water, and potential photosynthesis by associated phototrophs, will keep the aggregates well oxidized. If N₂ fixation takes place, it is likely carried out mostly by associated cyanobacterial diazotrophs (Farnelid et al., 2018; Klawonn et al., 2019). It may be speculated that aggregate associated cyanobacterial diazotrophs can switch to mixotrophic metabolism to sustain N₂ fixation as they sink to aphotic layers and photosynthesis is impaired (e.g., Rahav et al., 2013). Over time, aggregate-associated heterotrophic bacteria will proliferate, while preferentially exploiting labile nitrogen-rich organic compounds (Smith et al., 1992; Schneider et al., 2003), growing to cell concentrations commonly several orders of magnitude higher than in the surrounding water (Grossart and Simon, 1993; Turley and Mackie, 1994). This raises the C:N values of the aggregate over the Redfield ratio, gradually generating local nitrogen limitation (Figure 1C). At the same time extensive bacterial respiration exceeds the influx of oxygen diffusing from the surrounding water and causes formation of low oxygen microzones within the aggregate (Figure 1B). There is now a window of opportunity for N₂ fixation by NCDs fueled by aerobic respiration. The low oxygen microzones may become anoxic if extensive bacterial respiration continues and exceeds the diffusive oxygen flux into the particle from the surrounding environment. NCDs may then switch to anaerobic respiration using nitrate or sulfate as alternative electron acceptors to meet their energetic requirements, as has been described for other aggregate-associated microbial processes (Wright et al., 2012; Bianchi et al., 2018) and recently modeled for NCDs (Chakraborty et al., 2021). The usual inhibition of N₂ fixation by nitrate can be outweighed by enhanced diazotroph growth under low N:P ratio conditions (i.e., phosphate in excess; Knapp, 2012). However, it is unknown whether the high nitrate levels in deep waters may affect aggregate-associated NCD activity. It has been suggested that due to the high energetic costs associated with nitrate reduction, bacteria designed for diazotrophy may have few ecological reasons to use nitrate as a nitrogen source (Sprent and Sprent, 1990). In addition that high cell concentration near the surface of the aggregate may exhaust the supply of nitrate to the aggregate interior, supporting prevalence of sulfate over nitrate respiration within the aggregate (Chakraborty et al., 2021). Eventually, most of the labile carbon is consumed and heterotrophic respiration decreases. At that time, oxygen levels in the aggregate increase as oxygen consumption is exceeded by its diffusion from the surrounding water leading to significant reduction in N₂ fixation rates by NCDs.

This conceptual framework for the interaction between NCDs and the dynamic environment on aggregates was recently modeled and yielded N₂ fixation rates comparable to bulk rates measured in aphotic waters (Chakraborty et al., 2021), and agrees with field observations (Rahav et al., 2013, 2015; Benavides et al., 2016). Factors such as the level and type of substrate within the aggregate, the size of the aggregate, and its sinking speed may regulate the extent of aggregate associated N₂ fixation both directly or indirectly, as they modulate the placement of the aggregate within the vertical gradients of nitrate, oxygen and carbon in the water column (Klawonn et al., 2015; Bianchi et al., 2018; Chakraborty et al., 2021).

We argue that aggregates act as dynamic loci suitable for N₂ fixation by NCDs in aquatic ecosystems. Molecular analyses and microscopical identification have shown that material collected in large size fractions and sediment trap material harbor clusters of NCDs. However, the phylogeny, the specific N₂ fixation rates of NCDs on aggregates and their contribution to nitrogen cycling remain largely unquantified. It is, therefore, important to develop dedicated methods and approaches capable of isolating NCD-specific N₂ fixation rates and to identify their phylogeny. Our recommendation to the scientific community is to (1) develop cell-specific staining methods combined with ¹⁵N₂ labeling, (2) consider the 3D architecture of single aggregates, and (3) account for their heterogeneous spatiotemporal distribution in aquatic ecosystems. Advances in automated particle characterization and counting should increase the throughput of these methods in the future. These recommendations will inspire future research to unveil the ecology and quantitative relevance of aggregate-associated NCDs in marine as well as freshwater environments.

LR, ER, MB, and EB-Z wrote the manuscript. UP, H-PG, DB, IK, and ME commented on the final version of the manuscript. All authors contributed to the article and approved the submitted version.

MB and LR were supported by the BNP Paribas Foundation for Climate and Diversity grant “NOTION.” LR was supported by the Danish Council for Independent Research (6108-00013B). EB-Z was supported by the Israeli Science Foundation (grant number 944\21). H-PG was supported by the German Science Foundation (GR1540/28-1 and 37-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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