School of Engineering and Science - Jacobs University

Material and Methods
4.5.3 Quantification of pyoverdin by fluorescence measurement
The presence of pyoverdin was estimated by fluorescence measurements
using the F-2500 spectrofluorometer (Digilab, Krefeld). Purified pyoverdin of
Pss (courtesy of Prof. J.M. Meyer, Strasbourg, France) was used as standard.
For comparison between standard and sample, the pyoverdin was dissolved in
Pipes-medium (1 mg/ml). The excitation and emission spectrum was analysed
and an excitation maximum at λ= 397 nm as well as a maximum of emission at
λ= 458 nm was determined. Thus these parameters were applied for the
quantification measurements. A dilution series of pyoverdin in Pipes medium
served as equilibration curve for quantification. The fluorescence of the sample
was linear to pyoverdin concentration in a range of 0.5-7 µg pyoverdin/ml.
Culture supernatants were diluted until the fluorescence of the sample was in
the linear range.
4.5.4 Purification of siderophores by XAD4
Siderophore-containing supernatant of 5b cultures were harvested after 48 h of
incubation. The pH of the supernatant was subsequently adjusted to pH = 6 for
pyoverdin purification and to pH = 3 for purification of achromobactin or the
achromobactin-like siderophore. 50 ml to 150 ml of XAD4-resin (Sigma) were
loaded to a column. The column was pre-washed with water (1 L) and then the
supernatant was applied by gravity flow. Subsequently, the column was
washed with water (2 times the supernatant volume). Finally, the siderophore
was eluted in 100% methanol, until no more siderophore activity could be
measured in the eluate. The eluate was brought to dryness in a rotation
evaporator and eluted in 5-10 ml volume of water. This sample was brought to
dryness using a freeze drying apparatus (Edwards).
For fast screening of siderophores, this purification method was downscaled.
2 ml of culture supernatant were harvested and applied on a Mobicon minicolumn
(Mobitech, Göttingen) containing 2 ml of XAD4-resin. Supernatant was
applied to the column twice in order to improve siderophore binding. The
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