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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.


Sampling procedures. This figure shows the schematic proceedings for the applied sample extraction from culture broths.
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Figure 4: Sampling procedures. This figure shows the schematic proceedings for the applied sample extraction from culture broths.

Mentions: The quantifications of bio dry weight, oil and rhamnolipid contents were performed according to the extraction protocol shown in Figure 4. For oil and cell separation, 5 ml of cultivation broth was mixed with 5 ml of hexane (to separate the nonpolar compounds) and centrifuged at 7500 g at 15°C for 15 minutes. 200 μl of the lower, aqueous phase was used for the ATR – FTIR analysis, as described below. For HPLC reference analysis, 3 ml of the lower phase were transferred and acidified with 85% H3PO4 to pH = 2 – 3. 4 ml of ethyl acetate were added for the extraction of the rhamnolipids. After mixing, the suspension was centrifuged at 7500 g at 4°C for 15 minutes. 2 × 1.5 ml of the upper phase were transferred to 2 ml cups and the ethyl acetate was evaporated at 60°C in a thermo mixer for 24 h and processed as described in the HPLC section.


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

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

Sampling procedures. This figure shows the schematic proceedings for the applied sample extraction from culture broths.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sampling procedures. This figure shows the schematic proceedings for the applied sample extraction from culture broths.
Mentions: The quantifications of bio dry weight, oil and rhamnolipid contents were performed according to the extraction protocol shown in Figure 4. For oil and cell separation, 5 ml of cultivation broth was mixed with 5 ml of hexane (to separate the nonpolar compounds) and centrifuged at 7500 g at 15°C for 15 minutes. 200 μl of the lower, aqueous phase was used for the ATR – FTIR analysis, as described below. For HPLC reference analysis, 3 ml of the lower phase were transferred and acidified with 85% H3PO4 to pH = 2 – 3. 4 ml of ethyl acetate were added for the extraction of the rhamnolipids. After mixing, the suspension was centrifuged at 7500 g at 4°C for 15 minutes. 2 × 1.5 ml of the upper phase were transferred to 2 ml cups and the ethyl acetate was evaporated at 60°C in a thermo mixer for 24 h and processed as described in the HPLC section.

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.