Isolation, purification, and characterization of newDaphnia-toxic compound from axenicMicrocystis flos-aquae strain...

A new compound, that is toxic toDaphnia, called theDaphnia-toxic compound (DTC), was isolated and purified from water extracts ofMicrocystis PCC7806. An establishedDaphnia-bioassay was used as a detection system for the DTC in purified fractions. Temperature and pH stability were used to separate DTC from unstable compounds by changing the pH while the temperature was kept at 90°C. This was followed by ultracentrifugation at 100.000g. A molecular cutoff filtration technique was used to separate compounds with a molecular weight >100,000 Da and to reduce the volume of the fraction that was >500 Da. Evaporation in a vacuum led to a fraction that had a suitable volume for further characterization procedures. DTC was resistant to degradation by two bacteria that were isolated from a laboratory culture. DTC was not inactivated by incubation with protease K or pronase E. A strong anion-exchange resin at the stationary phase of the FPLC equipment was used to purify and concentrate the DTC. The molecular weight determined by gel filtration was between 1.0 and 3.1 kDa. A UV spectrum from this purified fraction exhibited absorption maxima at 220, 260, and 360 nm. The ecological and analytical relevance of the DTC is compared with microcystins.