It Takes Two to Fix and Transfer Nitrogen!

Diatoms are not able to acquire Nitrogen from N₂, therefore, they rely on dissolved fixed inorganic nitrogen pools which are made up of nitrates and ammonium. These pools are present at extremely low concentrations, so diatoms housing diazotrophs (which are nitrogen fixing bacteria) have an advantage. The diazotrophs fix the nitrogen and the diatoms acquire the fixed products through symbiosis. Symbiotic diatoms have been observed in the entire major ocean basins however, this paper looked at two regions of the Pacific Ocean. The symbiotic relationship they looked into suggests a primary interaction consists of fixed nitrogen being provided to a partner diatom by a potentially nitrogen fixing symbiont.
This paper looked at several heterocystous cyanobacteria, and their partner diatom genres. The species Richelia, which is a filamentous heterocystous cyanobacteria which can occupy Hemiaulus and Rhizosolenia. However, the other filamentous heterocystous cyanobacteria Calothrix only occupies Chaetoceros. These diatom-cyanobacteria symbionts have been understudied. The Richelia and Calothrix species have morphologically similar filaments called trichomes which consist of vegetative cells and heterocyst terminals. Heterocysts are specialised cells where nitrogen is fixed. The length, location and number of trichomes present in Richelia and Calothrix per diatom partner, and the phylogeny of symbionts differ in each symbiosis.
Estimates from previous nitrogen fixing studies were from bulk water assays or cell concentrations. These estimates included fixation from co-existing populations of Trichodesmium, a unicellular cyanobacteria. This paper is the first experimental evidence of nitrogen fixation and transfer of nitrogen products in field populations.

There were 2 sets of long term incubation (0, 24, 48 and 76 hours) experiments in July 2008, and 1 short term experiment (0 & 30 minutes, 1, 3 and 12 hours) in July 2009. The seawater used was collected from a depth of 0-10m using a CTD (conductivity, temperature, depth) rosette.
In July 2008, two experimental designs were used:

1. 4.4l bottles of seawater from the CTD, capped, sealed (without air bubbles) and then subsequently amended with 3ml of N.
2. 5l bottles of filtered seawater, all cell diameters above 5µm were incubated (all incubations were at ambient sea surface temperature) with 200ml of filtered seawater, capped, sealed (without air bubbles), and then amended with 150µl of N.

In July 2009, only the 4.4l bottles were used, and the filters were 3µm in pore size. In July 2008, the filter pore size was 3 or 10µm. All filters were washed 3 times in 0.1M phosphate=buffered saline and stored dry ad -20°C.

Next, nano-SIMS (Nanometre scale secondary ion mass spectrometry) analyses were used to measure the ratio of ¹⁵N/¹⁴N.

All cells were identified by their shape, cell diameter and excitation patterns, which were looked at using a an epi-fluorescent microscope fitted with blue and green excitation filters. The taper of the trichome was also used to distinguish between Richelia and Calothrix symbionts. Regions of interest (ROI) were defined around cells by epi-fluorescent images from before and after the nano-SIMS, and for each ROI, the ratios of ¹⁵N/¹⁴N were calculated.

Carbon content from the diatoms and the symbionts was calculated using Strathmann (1967) equations, and this carbon content was then used to estimate the nitrogen content using a method from a paper by Redfield in 1934.

The equations used to determine nitrogen assimilation, growth rate, specific growth rate etc are (hopefully) simplified above.

The results showed that the cyanobacterial partners were fully supporting the diatom Nitrogen requirements for growth, they observed equal or higher enrichment in the diatoms than in the vegetative cells. 

The nitrogen enrichment pattern was illustrated within a chain of Hemiaulus cells via electron microscopy, this mirrored the location of two associated Richelia trichomes which showed that the cyanobacteria had fixed the nitrogen. Areas within the Hemiaulus cholorplasts were also found to be enriched, this suggests that the nitrogen was transferred from symbiont to diatom partner.

Hotspots were identified in the species Crocosphaera associated with the diatom Climacodium, this diatom-cyanobacterial symbiosis is less studied, and has not been previously demonstrated hence the exact location of the symbiosis is still unknown.

The nanoSIMS anaylysis results prove that the N₂ fixing cyanobionts provide Nitrogen to their diatom counterparts. the enrichment was high in long term experiments after 24 hours, however they were unable to estimate the nitrogen transfer over a shorter time interval.

The short term experiments showed elevated nitrogen enrichment in Hemiaulus cells incubated for as short as 30min similar to cells measured after 3hours and 12hours incubations.

They expected the nitrogen enrichment to be slow because symbionts reside externally to the diatom cell membrane.

¹⁵N/¹⁴N ratios were determined on the individual symbiotic cells by nanoSIMS and were also used to estimate growth rates for all three symbionts cell types and their diatom partners.

The growth rates for Hemiaulus were similar to those reported for laboratory cultures of Rhizosolenia clevi which associates with a closely related Richelia strain of Hemiaulus. 

The other two symbionts, including the unicellular Crocosphaera cells associated with Climacodium, were also similar to the growth rates of Richelia, which was equally surprising given Rhizosolenia celvi's size (Rhizosolenia celvi are much larger in cell length) and the differing cell types of symbionts (unicellular and heterocystous). The growth rates for symbiont and diatom partners were so similar that a synchronous division would be expected.

The Richelia symbionts fix between 71-651% more nitrogen than required for their own growth. Trichodesmium also fix more nitrogen than required for their own growth. Due to the energy cost and high regulation, the authors suspect that the host diatoms may influence the nitrogen metabolism of the symbionts. They based this theory on the fact that terrestrial cyanobionts undergo structural-functional changes, which is co-ordinated by host to maximize nutrient transfer and balance growth, this scenario may exist in marine symbioses.

To conclude, symbiotic diatom populations could be an equally important source of new nitrogen, like the free-living colonial diazotrph trichodesmuim which is largely responsible for nitrogen fixation  in open oceans. Due to the inability to measure the nitrogen release from symbioses, estimates are more likely to be an underestimation. Research now says that nitrogen fixation rates will be underestimated when the ¹⁵N₂ tracer is introduced, having a knock on effect on the original estimations.

Interpreting the paper, it can be suggested that the more diatoms present may mean more diatom-cyanobacterial symbionts forming, this will increase the amount of Nitrogen fixed in the open ocean because the symbionts have a greater rate of nixtrogen fixation than free-living cyanobacteria, so both host and symbiont benefit.

Joint post by Kathryn Kavanagh & Hannah Prentice.

Foster, R.A et al (2011). Nitrogen Fixation and Transfer in Open Ocean Diatom-Cyanobacterial Symbioses. ISME. p1484-1493.