Driven by a building debate in the scientific world, regarding the discrepancy of N sources and sinks, Foster R.A et al decided to study the N2-fixing events of diatoms; mainly the genera Hemiaulus, Climacodium and Chaetoceros, and their symbiotically related heterocystous, filamentous cyanobacteria; Richelia intracellularis, Crocosphaera watsonii and Calothrix rhizosoleniae. These symbiotic diatoms have been observed internationally in blooms but N2 and C fixation rates have never been monitored. Previously, no advantage of the relationship had been identified but it was hypothesized that the cyanobacteria were providing their hosts with fixed N.
Long-term incubation of cultures from the Gulf of California and the sub-tropical North Pacific were performed and nanometer scale secondary ion mass spectrometer (NanoSIMS) was used to provide evidence that the fixed N2 was being transferred to the symbiotic diatoms.
Diatoms are unable to obtain N from N2 and so, they rely on dissolved inorganic nitrogen in the form of nitrate and ammonium, which exists at extremely low concentrations in the open ocean. Within Hemiaulus, NanoSIMS was employed to show the quantifiable levels of the 15N label in each sample where, using epifluroescence, symbiotic diazotrophs were shown to be residing. This verified that R. intracellularis was fixing N from N2. However, the diatoms’ chloroplasts were also enriched with N signifying that it was transferred from the symbiotic companion.
The 15N label was most apparent in the cyanobacteria, C. watsonii associated with Climacodium. In Chaetoceros, the observed C. rhizosoleniae were also shown to be acquiring high-levels of N2; this was especially abundant in the heterocyst and vegetative cells suggesting that the N was shifting from the C. rhizosoleniae, along the trichome and across the cell membrane of Chaetoceros. This was also observed in the symbiotic relationship between C. watsonii and Climacodium. These were previously unrecorded function.
It is interesting to note that in all cases studied, there was an equal or higher enrichment of N in the diatoms than in the vegetative cells. Diatoms in oligotrophic conditions were thought to grow slowly due to the low concentrations of nutrients. However, the team discovered that enrichment of the 15N label was saturated after 3hrs, faster than N-fixation was originally anticipatedin cyanobacteria.
The transfer of the N was also surprising. It was thought to be slower due to the placement of the cyanobacteria strains. R. intracellularis is located between the frustule and the cell membrane, C. watsonii location is unknown and the C. rhizosoleniae is located extracellularly. The rapid transfer of the N was due to the observed movement of N through the trichomes and the cell membrane (which was previously unobserved).
The growth rates of the diatoms and their symbionts were all found to be very similar. But a difference in the growth rate of free-living C. rhizosoleniae and R. intracelllaris, and those cyanobacteria symbiotically existing with their hosts; the former having a much slower growth rate and a smaller terminal size. The free-living cyanobacteria also showed slower N-fixation rates.
These relationships had always been assumed with little evidence, however, this paper clearly demonstrates the hypothesis that the cyanobacteria symbionts fully support the diatom’s need of N for cell growth and is significant enough for symbiotic diatoms to be included in N-fixation models.