Microcystin Extraction

Assessment of extraction methods for microcystin-LR from Microcystis aeruginosa.

Method

Cultured Microcystis aeruginosa cells were filtered through preweighed glass fibre filters (GF/C 47 mm dia. - Whatman) and dried in a vacuum desiccator overnight at room temperature (approx. 22 degrees C). Filters were reweighed the following morning and extracted in one of five extraction solvents for assessment (2 filters per solvent, 2.0 ml of solvent per filter). The solvents used were as follows;

₂

(2) 5% Acetic acid : 95% Milli-Q H₂O

(3) 50% Methanol : 50% Milli-Q H₂O

(4) 100% Milli-Q H₂O

(5) 5% n-Butanol : 20% Methanol : 75% Milli-Q H₂O

Filters were extracted for one hour with shaking and 1.0 ml of each extract was filtered for analysis by HPLC. The remaining liquid in each tube was discarded and an additional 2.0 ml of the appropriate solvent added to each of the filters. Again, extraction was for one hour with shaking. The proceedure was repeated until each filter had been extracted three times in 2.0 ml of one of the five solvents.

For the sake of simplicity (and the absence of more microcystin standards) the extraction of microcystin-LR by each solvent was analysed. The results are summarised in the tables below.

Percent of microcystin-LR extracted per extraction using the solvents listed above

		Extraction
Solvent	1st	2nd	3rd
80% Ethanol	100	0	0
5% Acetic acid	0	0	0
50% Methanol	84.6	10.4	5.0
100% H₂O	82.9	12.8	4.3
n-Butanol:Methanol:Water	82.2	13.0	4.8

Relative microcystin-LR extraction efficiency of each solvent compared to n-Butanol:Methanol:Water (100%). That is, the total amount of microcystin-LR extracted using each solvent, relative to the amount extracted using n-Butanol:Methanol:Water.

Solvent	Extraction efficiency (% But:Meth:H₂O)
80% Ethanol	31.9
5% Acetic acid	0.0
50% Methanol	98.7
100% H₂O	77.4
n-Butanol:Methanol:Water	100

It is interesting to note that 80% Ethanol extracted only two major compounds from the strain of M. aeruginosa used in this experiment. Water extracted three major compounds, whereas the Butanol:Methanol:Water mix extracted four major peaks and 50% Methanol, five. (I hope to scan in a few chromatograms of each extraction solvent for you all to see... stay tuned).

Any comments, please e-mail me.

Ben Long
School of Botany
La Trobe University