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Fast quantitative determination of microbial rhamnolipids from cultivation broths by ATR-FTIR Spectroscopy.

Leitermann F, Syldatk C, Hausmann R - J Biol Eng (2008)

Bottom Line: Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy.An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research University Karlsruhe, Institute of Engineering in Life Sciences, Section of Technical Biology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Rudolf.Hausmann@tebi.uni-karlsruhe.de.

ABSTRACT

Background: Vibrational spectroscopic techniques are becoming increasingly important and popular because they have the potential to provide rapid and convenient solutions to routine analytical problems. Using these techniques, a variety of substances can be characterized, identified and also quantified rapidly.

Results: The rapid ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy) in time technique has been applied, which is suitable to quantify the concentrations of microbial rhamnolipids in a typical cultivation process. While the usually applied HPLC analysis requires an extensive and time consuming multi step extraction protocol for sample preparation, the ATR-FTIR-method allows the quantification of the rhamnolipids within 20 minutes. Accuracies between 0.5 g/l - 2.1 g/l for the different analytes were determined by cross validation of the calibration set. Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.

Conclusion: ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy. An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

No MeSH data available.


Validation of the developed PLS based quantification procedure of rhamnolipids. Results for the predictive quality of the performed cross validations and test set validation for rhamnolipid 1 (RL 1), rhamnolipid 3 (RL 3) and rhamnolipid total (RL total).
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Figure 3: Validation of the developed PLS based quantification procedure of rhamnolipids. Results for the predictive quality of the performed cross validations and test set validation for rhamnolipid 1 (RL 1), rhamnolipid 3 (RL 3) and rhamnolipid total (RL total).

Mentions: To establish multivariate calibration methods, a spectra set of about 80 samples, derived from 9 different rhamnolipid fermentations, was recorded. Using this spectra set, as a first step, three independent calibrations for rhamnolipid 1 and rhamnolipid 3, as well as the combined rhamnolipid 1 and 3 content, were set up. All calibrations were evaluated and optimized by cross validation, according to their determination coefficients R2, RMSECV (root mean square error of cross validation) and utilized PLS factors (synonym to the terminus rank). The results for the reference versus the predicted values are displayed in Figure 3. By an applied rank of five, similar determination coefficients R2 from 95.56 to 96.07 were calculated for all three analytes. For rhamnolipid 1 and rhamnolipid 3 a RMSECV of 0.496 g/l respectively 1.49 g/l was determined. For the total rhamnolipid 1 and 3 in the samples, an error of 2.06 g/l was found. For the calibration purposes of all three analytes, a single frequency area from 900 – 1200 cm-1 was selected. As mathematical pre-treatment for rhamnolipid 1 and rhamnolipid 3, the first derivative with the subtraction of a straight line and for rhamnolipid total the first derivative results in the highest determination coefficients and lowest prediction errors.


Fast quantitative determination of microbial rhamnolipids from cultivation broths by ATR-FTIR Spectroscopy.

Leitermann F, Syldatk C, Hausmann R - J Biol Eng (2008)

Validation of the developed PLS based quantification procedure of rhamnolipids. Results for the predictive quality of the performed cross validations and test set validation for rhamnolipid 1 (RL 1), rhamnolipid 3 (RL 3) and rhamnolipid total (RL total).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572035&req=5

Figure 3: Validation of the developed PLS based quantification procedure of rhamnolipids. Results for the predictive quality of the performed cross validations and test set validation for rhamnolipid 1 (RL 1), rhamnolipid 3 (RL 3) and rhamnolipid total (RL total).
Mentions: To establish multivariate calibration methods, a spectra set of about 80 samples, derived from 9 different rhamnolipid fermentations, was recorded. Using this spectra set, as a first step, three independent calibrations for rhamnolipid 1 and rhamnolipid 3, as well as the combined rhamnolipid 1 and 3 content, were set up. All calibrations were evaluated and optimized by cross validation, according to their determination coefficients R2, RMSECV (root mean square error of cross validation) and utilized PLS factors (synonym to the terminus rank). The results for the reference versus the predicted values are displayed in Figure 3. By an applied rank of five, similar determination coefficients R2 from 95.56 to 96.07 were calculated for all three analytes. For rhamnolipid 1 and rhamnolipid 3 a RMSECV of 0.496 g/l respectively 1.49 g/l was determined. For the total rhamnolipid 1 and 3 in the samples, an error of 2.06 g/l was found. For the calibration purposes of all three analytes, a single frequency area from 900 – 1200 cm-1 was selected. As mathematical pre-treatment for rhamnolipid 1 and rhamnolipid 3, the first derivative with the subtraction of a straight line and for rhamnolipid total the first derivative results in the highest determination coefficients and lowest prediction errors.

Bottom Line: Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy.An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research University Karlsruhe, Institute of Engineering in Life Sciences, Section of Technical Biology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Rudolf.Hausmann@tebi.uni-karlsruhe.de.

ABSTRACT

Background: Vibrational spectroscopic techniques are becoming increasingly important and popular because they have the potential to provide rapid and convenient solutions to routine analytical problems. Using these techniques, a variety of substances can be characterized, identified and also quantified rapidly.

Results: The rapid ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy) in time technique has been applied, which is suitable to quantify the concentrations of microbial rhamnolipids in a typical cultivation process. While the usually applied HPLC analysis requires an extensive and time consuming multi step extraction protocol for sample preparation, the ATR-FTIR-method allows the quantification of the rhamnolipids within 20 minutes. Accuracies between 0.5 g/l - 2.1 g/l for the different analytes were determined by cross validation of the calibration set. Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.

Conclusion: ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy. An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

No MeSH data available.