Figure 1.
Scopulariopsis brevicaulis mutant strains on WSP30 petri dishes; a) Example of UV treated (left) and not treated (right) conidia growing on petri dishes: UV-radiation was performed using 312 nm wavelength for a time periods of 85 seconds and 13.742 W m−2 (85 s: 1.17 kJ m−2) radiation intensity. 1% (mean of 20 colonies) of the conidia were germinating; b) Colonies of different mutant strains growing on petri dishes upon UV mutagenesis.
Figure 2.
Calibration curve of scopularide A (ScoA) performed with micrOTOF II, Bruker Daltonics; a) Comparison of measured and theoretical peak area of scopularide A concentrations: (− −♦− − (dark grey)) measured peak area (injection volume 10 µL), (–×– (light grey)) theoretical peak area (based on linear regression I); b) Linear part of the graph: (–♦– (dark grey)) measured peak area, (− −×− − (red)) measured peak area, below the linear correlation (injection volume 10 µL), (–– (light grey)) linear regression y = 4×108× x + 4×106, R2 = 0.9988 background noise of .
Linear area for calibration purposes was set to a range of to
(peak area), equivalent to a scopularide A concentration of 0.008 to 0.125 mg mL−1.
Figure 3.
Comparison of standardized and optimized method: The miniaturization and optimization of the screening procedure was set up in three parts, starting with the initial establishing of the UV-mutant library.
The cultivation in 24-well plates (upstream), the extraction (downstream) and the quantitative analysis including the sample preparation for LC-MS of scopularide A were compared separately to the standardized screening procedure in 300 mL Erlenmeyer flasks containing 100 mL medium.
Figure 4.
Spiking addition of scopularide A (ScoA), to determinate extraction comparability, results for 0.1 and 0.2 mg mL−1 are shown (scopularide A per biomass [mg mL−1 per g] of cultures plus spiking (dark grey) and scopularide A per biomass [mg mL−1 per g] of pure cultures (light grey)).
Figure 5.
Comparison of relative error of measured values of both linear regression I (: − −•− − (light grey)) and II (
: –▴– (dark grey)) in dependence of scopularide A (ScoA) concentration [mg mL−1] given for the measured values of the calibration curve (injection volume 10 µL).
Linear regression II containing a background of showed a much more defined agreement of the measured and calculated values within the linear area, as a distinct plateau was visible. Therefore a background noise was accepted for the calculation of the scopularide A concentrations.
Table 1.
Comparison of arithmetical mean and median of scopularide A production for 1