Single cell dynamics and nitrogen transformations in the chain forming diatom Chaetoceros affinis

Colony formation in phytoplankton is often considered a disadvantage during nutrient limitation in aquatic systems. Using stable isotopic tracers combined with secondary ion mass spectrometry (SIMS), we unravel cell-specific activities of a chain-forming diatom and interactions with attached bacteria. The uptake of 13C-bicarbonate and15N-nitrate or 15N-ammonium was studied in Chaetoceros affinis during the stationary growth phase. Low cell-to-cell variance of 13C-bicarbonate and 15N-nitrate assimilation within diatom chains prevailed during the early stationary phase. Up to 5% of freshly assimilated 13C and 15N was detected in attached bacteria within 12 h and supported bacterial C- and N-growth rates up to 0.026 h−1. During the mid-stationary phase, diatom chain-length decreased and 13C and 15N-nitrate assimilation was significantly higher in solitary cells as compared to that in chain cells. During the late stationary phase, nitrate assimilation ceased and ammonium assimilation balanced C fixation. At this stage, we observed highly active cells neighboring inactive cells within the same chain. In N-limited regimes, bacterial remineralization of N and the short diffusion distance between neighbors in chains may support surviving cells. This combination of “microbial gardening” and nutrient transfer within diatom chains represents a strategy which challenges current paradigms of nutrient fluxes in plankton communities.


Supplementary figures
Fig. S1 NO3 -concentration (blue triangles), PON estimated from plate reader data continuously calibrated against manual cell count to account for changes in cellular pigment abundance (black circles).Vertical lines show sampling times after 13 DIC and 15 NO3 -: solid line: T0, dashed line: 12 h L and dotted line: 12:12 h L:D, for the early, mid, and late stationary growth phase, day 9, 15 and 21 respectively.Numbers on the x axis represent 15:00 during each day.The error bars represent SE.

Early stationary
Mid stationary Late stationary  Table S1 Dissolved   Number of analyzed cells: Fig. S2 DIC and NO3 -assimilation by individual C. affinis cells in chains during the early stationary phase, as a function of the observed cells position relative to one end of the chain.Each line/color represents one chain, each symbol represents an individual cell, Circles: cells found at the end of a chain, Squares: cells not in the end of the chain, Triangles: cell position could not be determined, A, C & E: after 12h L, B, D & F: after 12:12h L:D, A & B: DIC assimilation by individual C. affinis cells in chains.C & D: NO3 -assimilation by individual C. affinis cells in chains.E & F: DIC:NO3 - assimilation ratio, dashed lines represent the Redfield C:N ratio.

NO3
Free living bacteria, assimilation of diatom derived C and NDiatom derived 13 C-DIC transferred to free-living bacteria (fmol bacteria -1 ) 100 76 41Diatom derived 15 N-NO3 -transferred to free-living bacteria (fmol bacteria -1 ) 100 11 0 nutrient concentrations (average value ± SE for 3 replicates measured directly after isotopic tracer additions).The percentages represent NH4 + contribution to dissolved bioavailable DIN and the relative contribution to diatom assimilation in the last two columns respectively.TableS2Assimilation of C and N by free-living and diatom attached bacteria, bacterial abundance, and transfer rates per diatom.Each value represents the mean ± SE.

Table S3
Diatom abundance, POC, PON, total DIC and DIN assimilation and DIC and DIN assimilation measured in C.affinis by SIMS.Each value represents the mean value, ± SE.

Table S4
Average chain length and cell size during each growth phase ± SE, n = 9.Observations were made using light microscopy.TableS5Measured C and N content cell -1 and estimated content usingRedfield 1934, Menden-Deuer and Lessard 2000, Sun 2003.During the early mid and late stationary growth phase here shortened to E-STAT, M-STAT, and L-STAT respectively.Each value represents means ± SD.Bold values are averages for the entire incubation.

Table S6 A
compilation of measurements, shown as percentages (%) relative to the value in the early stationary growth phase.

Table S7 C
-and N-specific growth rates for diatom attached and free-living bacteria.Each value represents the mean value ± SE.Numbers in brackets represents the highest and lowest positive observation.