Towards establishment of axenic cyanobacteria cultures

In the EU FP7 project MAREX (www.marex.fi), bioactive properties of various marine organisms are studied. In the framework of the project, rock pool cyanobacteria were collected from the Åland Islands, Finland, identified and monoalgal cultures established. Axenic algae cultures are crucial when exploring bioactivity, as they provide certainty that the relevant compounds are actually produced by the algae and not associated bacteria (Enss & Rischer 2012). A protocol modified from Su et al. (2007) was tested for production of axenic cultures of cyanobacteria. The cyanobacteria were cultured for two weeks (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1) in Z8+N growth medium (Spoof & Meriluoto 2005). In order to fragment the filamentous cyanobacteria, steel marbles (ø 4 mm) were added to 25 ml cultures, followed by vortexing in periods of 10 s (max. three periods). Cells were harvested by sequential filtration (5, 20, 80 µm pore size), washed three times in 50 ml Z8+N (2000 * g, 5 min), and incubated in 50 ml wash medium (Z8+N, 0,1 M EDTA, 0,0005 % Tween-20) for 1 h at 24 °C. Lysozyme (1.2 g/l, or 0.8 g/l) was added, the cells incubated for 10 min, washed two times (2000 * g, 5 min) and finally resuspended in Z8+N. Antibiotics (timentin 150 µg/ml, gentamycin 100 µg/ml, cefotaxime 250 µg/ml, ampicillin 250 µg/ml, meropenem 10 µg/ml) were added to the algal suspensions. The cultures were incubated 24 h in dark conditions and subsequently in light (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1). Antibiotics were removed from aliquots of the cultures after one and three weeks. The axenity of the cultures was tested by plating of bacteria on Difco Marine Agar 2216. After three week incubation, no bacterial growth was observed. A method for confirmation of the axenity of viable cyanobacteria cultures will be developed. So far the cyanobacterial cells appear intact, but growth was not noticed in the aliquots. The viability of the cells will be tested by fluorescence staining using methods comparable to those used by Sato et al. (2004) and de los Ríos et al. (2004). According to our results, the employed method is not directly suitable for establishment of axenic cyanobacteria cultures. However, our work showed that the tested cyanobacteria can resist short incubation in quite high lysozyme concentration, while a short incubation in SDS, even in low concentration (final concentration 0.005 %), bleached the cells instantly.