Isolation, growth, and nitrogen fixation rates of the (diatom-cyanobacterium) symbiosis in culture.

Nitrogen fixers (diazotrophs) are often an important nitrogen source to phytoplankton nutrient budgets in N-limited marine environments. Diazotrophic symbioses between cyanobacteria and diatoms can dominate nitrogen-fixation regionally, particularly in major river plumes and in open ocean mesoscale blooms. This study reports the successful isolation and growth in monocultures of multiple strains of a diatom-cyanobacteria symbiosis from the Gulf of Mexico using a modified artificial seawater medium. We document the influence of light and nutrients on nitrogen fixation and growth rates of the host diatom Grunow together with its diazotrophic endosymbiont Schmidt, as well as less complete results on the - symbiosis. The symbioses rates reported here are for the joint diatom-cyanobacteria unit. Symbiont diazotrophy was sufficient to support both the host diatom and cyanobacteria symbionts, and the entire symbiosis replicated and grew without added nitrogen. Maximum growth rates of multiple strains of symbioses in N-free medium with N as the sole N source were 0.74-0.93 div d. Growth rates followed light saturation kinetics in symbioses with a growth compensation light intensity (E) of 7-16 µmol msand saturation light level (E) of 84-110 µmol ms. Nitrogen fixation rates by the symbiont while within the host followed a diel pattern where rates increased from near-zero in the scotophase to a maximum 4-6 h into the photophase. At the onset of the scotophase, nitrogen-fixation rates declined over several hours to near-zero values. Nitrogen fixation also exhibited light saturation kinetics. Maximum N fixation rates (84 fmol N heterocysth) in low light adapted cultures (50 µmol ms1) were approximately 40-50% of rates (144-154 fmol N heterocysth) in high light (150 and 200 µmol ms) adapted cultures. Maximum laboratory N fixation rates were ~6 to 8-fold higher than literature-derived field rates of the symbiosis. In contrast to published results on the symbiosis, the symbiosis did not use nitrate when added, although ammonium was consumed by the symbiosis. Symbiont-free host cell cultures could not be established; however, a symbiont-free strain was isolated directly from the field and grown on a nitrate-based medium that would not support DDA growth. Our observations together with literature reports raise the possibility that the asymbiotic are lines distinct from an obligately symbiotic line. While brief descriptions of successful culture isolation have been published, this report provides the first detailed description of the approaches, handling, and methodologies used for successful culture of this marine symbiosis. These techniques should permit a more widespread laboratory availability of these important marine symbioses.