Establishment of axenic cultures from cyanobacterium Aphanizomenon flos-aquae akinetes by micromanipulation and chemical treatment

Filamentous cyanobacteria are an environmentally and biotechnologically important species. However, the isolation and purification techniques for these organisms remain poorly developed and rarely used in studies of their biology, ecology and growth requirements, mainly due to the need of species- or strain-specific approaches and labour-intensive work. Here we propose a simple protocol for the establishment of an axenic (pure) culture of filamentous cyanobacterium Aphanizomenon flos-aquae from its akinetes. We tested the effect of different physical and chemical treatments on akinetes viability and germination time, removal of epiphytic and contaminating bacteria and growth of the recovered cultures. The protocol consists of three steps: 1) capturing the A. flos-aquae akinetes using a micromanipulator, 2) akinete treatment with a TESC buffer, containing 1% of CTAB and 3) transferring the akinetes to the growth medium. We further demonstrate the increased growth of axenic A. flos-aquae compared to their bactericized counterparts, which provides insights into cyanobacteria-bacteria interactions.