Utilization of ammonium by the hydrocarbon-producing microalga, Botryococcus braunii Showa

- Botryococcus braunii Showa was cultured with ammonium as the nitrogen source.
- Toxicity of ammonium was ameliorated with buffered media.
- Hydrocarbon production was not altered under ammonium nutrition with buffered media.
- A cDNA clone of a functional ammonium transporter was isolated from B. braunii Showa.

As a prospective feedstock for sustainable biofuels, the green microalga Botryococcus braunii race B has attracted significant attention since this colony-forming alga has the ability to accumulate considerable amounts of triterpene hydrocarbons, botryococcenes, in its extracellular matrix. However, its primary metabolism, nutrient uptake and utilization relevant to growth, are still poorly understood in this alga. From this perspective, we investigated the utilization of ammonium by B. braunii as a more reduced form of nitrogen source compared to nitrate, which is generally used for cultures of the alga. Isotope-ratio mass spectrometry analyses indicated that the uptake rate of ammonium was higher than that of nitrate at a certain concentration. When ammonium was added to culture medium, however, it inhibited algal growth even in the presence of nitrate. Buffered culture media ameliorated the toxicity of ammonium, and the alga accumulated the same levels of hydrocarbons and secondary carotenoids, irrespective of nitrogen source. To characterize utilization of ammonium by B. braunii at the molecular level, putative ammonium transporter (AMT) genes were screened from a cDNA library and four isogenes (BbAMT1;1, 1;2, 1;3 and 1;4) were cloned. Two of them, BbAMT1;1 and 1;2, were upregulated under nitrogen deficient conditions. BbAMT1;1 complemented the growth of an ammonium uptake-defective yeast strain, showing it to be a functional gene coding for AMT. These results could pave the way for culturing B. braunii more efficiently using ammonium as a sole nitrogen source by improving ammonium uptake through our understanding of AMT.