The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses
Abundances of N2 fixing symbioses in the WTNATo date, the various marine symbiotic diatoms are notoriously understudied, and hence our understanding of their abundances and distribution patterns is limited [7]. In general, these symbiotic populations are capable of forming expansive blooms, but largely co-occur at low densities in tropical and subtropical waters with a few rare reports in temperate waters [26,27,28,29, 39,40,41,42]. The Rhizosolenia-Richelia symbioses have been more commonly reported in the North Pacific gyre [26, 27, 31], and the western tropical North Atlantic (WTNA) near the Amazon and Orinoco River plumes is an area where widespread blooms of the H. hauckii-Richelia symbioses are consistently recorded [28, 29, 42,43,44,45,46,47].In the summer of 2010, bloom densities (105â106 cells Lâ1) of the H. hauckii-Richelia symbioses were encountered at multiple stations with mesohaline (30â35 PSU) surface salinities (Supplementary Table 1). The R. clevei-Richelia symbioses were less abundant (2â30âcellsâLâ1). Similar densities of H. hauckii-Richelia have been reported in the WTNA during spring (AprilâMay) and summer seasons (JuneâJuly) (28â29; 46). In fall 2011, less dense symbiotic populations (0â50âcellsâLâ1) were observed, and the dominant symbioses was the larger cell diameter (30â50â”m) H. membranaceus associated with Richelia. Previous observations of H. membranaeus-Richelia in this region are limited and reported as total cells (i.e., 12-218 cells) and highest numbers recorded in AugâSept in waters near the Bahama Islands [43]. On the other hand, Rhizosolenia-Richelia are even less reported in the WTNA, and most studies by quantitative PCR assays based on the nifH gene (for nitrogenase enzyme for N2 fixation) of the symbiont (44; 46â7). Unlike qPCR which cannot resolve if the populations are symbiotic or active for N2 fixation, the densities and activity reported here represent quantitative counts and measures of activity for symbiotic Rhizosolenia.The WTNA is largely influenced by both riverine and atmospheric dust deposition (e.g., Saharan dust) [48], including the silica necessary for the host diatom frustules, and trace metals (e.g., iron) necessary for photosynthesis by both partners and the nitrogenase enzyme (for N2 fixation) of the symbiont. We observed similar hydrographic conditions (i.e., low to immeasurable concentrations of dissolved N, sufficient concentrations of dissolved inorganic P and silicates, and variable surface salinities; 22; 28â29; 40â47) as reported earlier that favor high densities of H. hauckii-Richelia blooms. Unfortunately our data is too sparse to determine if these conditions are in fact priming and favoring the observed blooms of the H.hauckii-Richelia symbioses in summer 2010, and to a lesser extent in the Fall 2011.A biometric relationship between C and N activity and host biovolumeThe diatom-Richelia symbioses are considered highly host specific [10, 11], however, the driver of the specificity between partners remains unknown. We initially hypothesized that host selectivity could be related to the N2 fixation capacity of the symbiont. Moreover, it would be expected that the larger H. membranaceus and R. clevei hosts which are ~2â2.5 and 3.5â5 times, respectively, larger in cell dimensions than the H. hauckii cells would have higher N requirements (Supplementary Table 2). In fact, recently it was reported that the filament length of Richelia is positively correlated with the diameter of their respective hosts [22]. Thus, to determine if there is also a size dependent relationship between activity and cell biovolume, the enrichment of both 15N and 13C measured by SIMS was plotted as a function of symbiotic cell biovolume.Given the long incubation times (12âh) and previous work [32] that show fixation and transfer of reduced N to the host is rapid (i.e., within 30âmin), we expected most if not all of the reduced N, or enrichment of 15N, to be transferred to the host diatom during the experiment (Fig. 1). Therefore, we measured and report the enrichment for the whole symbiotic cell, rather than the enrichment in the individual partners (Supplementary Table 2; Fig. 2). The enrichment of both 13C/12C and 15N/14N was significantly higher in the larger H. membranaceus-Richelia cells (atom % 13C: 1.5628â2.0500; atom % 15N: 0.8645â1.0200) than the enrichment measured in the smaller H. hauckii-Richelia cells (atom % 13C: 1.0700â1.3078; atom % 15N: 0.3642â0.7925) (Fig. 2) (13C, MannâWhitney pâ=â0.009; 15N, MannâWhitney pâ50 symbiotic cells in a chain) were reported at station 2 with fully intact symbiotic Richelia filaments (2â3 vegetative cells and terminal heterocyst), and at station 25 chains were short (1â2 symbiotic cells) and associated with short Richelia filaments (only terminal heterocyst). Moreover, the symbiotic H. hauckii hosts possessed poor chloroplast auto-fluorescence at station 25 [46]. Given that the cells selected for NanoSIMS were largely single cells, rather than chains, we suspect that these cells were in a less than optimal cell state, which was also reflected in the low 13C/12C enrichment ratios and low estimated C-based growth rates (0.30â57 div dâ1). These are particularly reduced compared to the growth rates recently reported for enrichment cultures of H. hauckii-Richelia (0.74â93 div dâ1§) (Supplementary Table 2) [33].In 2011, higher cellular N2 fixation rates (15.4â27.2âfmols N cellâ1 hâ1) were measured for the large cell diameter H. membranaceus-Richelia, symbioses. Despite high rates of fixation, cell abundances were low (4â19 cells Lâ1), and resulted in a low overall contribution of the symbiotic diatoms to the whole water N2 ( >1%) and C-fixation ( >0.01%). The estimated C-based growth rates for H. membranaceus were high (1.9â3.5 div dâ1), whereas estimated N-based growth rates (0.3â4 div dâ1) were lower than previously published (33; 52â53). Hence the populations in 2011 were likely in a pre-bloom condition given the low cell densities.Estimating symbiotically derived reduced N to surface oceanTo date, determining the fate of the newly fixed N from these highly active but fragile symbiotic populations has been difficult. Thus, we attempted to estimate the excess N fixed and potentially available for release to the surround by using the numerous single cell-specific rates of N2 fixation determined by SIMS on the Hemiaulus spp.-Richelia symbioses (Supplementary Materials). Because the populations form chains during blooms and additionally sink, we calculated the size-dependent sinking rates for both single cells and chains ( >50 cells). Initially we hypothesized that sinking rates of the symbiotic associations would be more rapid than the N excretion rates, such that most newly fixed N would contribute less to the upper water column (sunlit).The sinking velocities were plotted (Fig. 5) as a function of cell radius at a range (min, max) of densities and included two different form resistances (â
â=â0.3 and 1.5). As expected, the combination of form resistance and density has a large impact on the sinking velocity. For example, a H. hauckii cell of similar radius (10âÎŒm) and density (3300âkgâmâ3) but higher form resistance (0.3 vs. 1.5) sinks twice as fast at the lower form resistance (Fig. 5). This points to chain formation (e.g., increased form resistance) as a potential ecological adaptation to reduce sinking rates. Recently, colony formation was identified as an important phenotypic trait that could be traced back ancestrally amongst both free-living and symbiotic diatoms that presumably functions for maintaining buoyancy and enhancing light capture [22].Fig. 5: The influence of cell characteristics on estimated sinking velocity for symbiotic Hemiaulus spp.The range of diatom sinking speed predicted using the modified Stokes approximation for diatoms [74] and accounting for the symbioses (cylinders) having varying cell size characteristics (form resistance by altering chain length, density; Supplementary Table 4). Note that form resistance increases with chain length and that the longest chains would have sinking speeds less than 10âm dâ1.Full size imageThe concentration of fixed N surrounding a H. hauckii and H. membranaceus cell were modeled (Supplementary Materials; Supplementary Table 4; Fig. 6). First, the cellular N requirement (QN, mol N cellâ1) for a cell of known volume, V, as per the allometric formulation of Menden-Deuer and Lessard [71] is calculated by the following.$${{{{{{{mathrm{Q}}}}}}}}_{{{{{{{mathrm{N}}}}}}}} = (10^{ – 12}/12) times 0.76 ;times, {{{{{{{mathrm{V}}}}}}}}^{^{0.189}}$$
(1)
Fig. 6: The simplified case of diffusive nitrogen (N) exudate plumes for non-motile symbioses.The concentration of dissolved N (nmol Lâ1) is presented at of varying cell sizes (3â”m and 30â”m) for H. hauckii-Richelia (A and B, respectively) and H. membranaceus-Richelia (C and D, respectively) growing at specific growth rates of 0.4 dâ1 (dashed red lines) or 0.68 dâ1 (solid black lines). Exudation follows the same principle as diffusive uptake as per Kiorboe [72] in the absence of turbulence.Full size imageVolume calculations assume a cylindrical shape; whereas exudation assumes an equivalent spherical volume. Then, using published growth rates of 0.4 dâ1 and 0.68 dâ1 for the symbioses [52, 53], N uptake rate (VN) necessary to sustain the QN was determined. N loss was assumed to be a constant fraction (f) of the VN; this fraction was assumed to be 7.5% and 11% for H. hauckii and H. membranaceus, respectively, or the estimated excess N which was fixed given the assumed growth rate [31]. The excretion rate (EN) of the individual cells was then calculated as$${{{{{{{mathrm{E}}}}}}}}_{{{{{{{mathrm{N}}}}}}}} = {{{{{{{mathrm{fQ}}}}}}}}_{{{{{{{mathrm{N}}}}}}}}$$
(2)
The concentration of fixed N surrounding the cell (Cr) was iteratively calculated by the following:$${{{{{{{mathrm{C}}}}}}}}_{{{{{{{mathrm{r}}}}}}}} = {{{{{{{mathrm{E}}}}}}}}_{{{{{{{mathrm{N}}}}}}}}/(4pi * {{{{{mathrm{D}}}}}}* {{{{{mathrm{r}}}}}}_{{{{{mathrm{{x}}}}}}}) + {{{{{{{mathrm{C}}}}}}}}_{{{{{{{mathrm{i}}}}}}}}$$
(3)
The concentric radius (rx) as per KiĂžrboe [72] uses a diffusivity of N assumed to be 1.860âĂâ10â5âcm2âsecâ1 and the background concentration of N (Ci) is assumed to be negligible. Figure 5 presents the results for the two symbioses: H. membranaceus and H. hauckii at the two growth rates and as chains or singlets. Mean sinking rates for cells with a high form resistance (e.g., chains) are More
