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

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.

Figure 1

Rhamnolipid 3 IR-spectrum. ATR-FTIR spectrum of an aqueous solution (89 mg/mL) of rhamnolipid 3. The allocation of conspicuous absorption to corresponding characteristic group absorptions from literature was attempted.

Figure 2

Reproducibility of spectra measurements. This figure displays four spectra of two fermentation derived samples after double application. A comparison of the corresponding two spectra of each sample showed correlations of above 99.9%.

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).

Figure 4

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