Hi everybody,
I am trying to measure the quantity of residual alkane left in the medium after an incubation period.
The problem is that, on solid long alkanes as C24 tetracosane, the bacterial cells are attaching strongly to the alkane and I am not sure that my extraction step is working well.
Briefly, I extract C24 once, from 100µL of culture whit 30µL of hexane C6, by shaking at 250rpm for 20 min and 10 min rest for phase separation. I collect 1mL of the organic phase for GS-MS residual alkane analyses.
The problem is that the organic phase is like a gel (probably due to the polysaccharides and biosurfactants), flows very poorly, bacterial cells remains in the same structure shape as if they are still attach to the alkane.
I tried to separate the cell from the alkane priory to extraction, by centrifugation, sonication and heating, but nothing worked.
I also tried using some other solvents instead of hexane (Dichloromethane, Chloroform) but the results were the same.
From my analyses, in 2 weeks of incubation, the quantity of alkane seems to be completely removed from the culture medium. But I am not sure if the results are correct, or the alkane extraction step was inaccurate and the alkane is not biodegraded, just fiscal trapped by the cells.
I, also, have to measure the dry weight of the cell, during this experiment.
Because the alkane is solid, when I collect the cells I also collect the alkane. So the cells dry weight values are influenced by the alkane weight value.
Any suggestions whit what I can wash the cells to remove the alkane (C24, or C32) priory to measuring their weight?
I am attaching a few picture as well.
Forgot to mention that I am tasting an Actinobacteria strain for it’ ability to degrade different alkanes.
I am thinking of using Tween80 and incubate the samples with this surfactant for a few hours before extraction. Does anybody try this before?
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