Identification and quantification of biosurfactants produced by the marine bacterium Alcanivorax borkumensis by hyphenated techniques

A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium’s attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C6–C12) and degrees of unsaturation (0–1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-023-04972-5.

Table S1 Detailed characterization of the identified species from pyruvate cultivation of A. borkumensis based on MS/MS.Characterization was performed for the individual retention times of the double peaks, where MS/MS could be recorded.The fragments detected in negative ion mode are listed, and the conclusions for the composition and position of the fatty acyl groups are summarized  Table S3 Detailed characterization of the identified species from hexadecane cultivation of A. borkumensis based on MS/MS.

Species
Characterization was performed for the individual retention times of the double peaks, where MS/MS could be recorded.The fragments detected in negative ion mode are listed, and the conclusions for the composition and position of the fatty acyl groups are summarized

MS/MS spectra for all identified species in negative ion mode (raw data).
For the different m/z, the EIC extracted with boxcar smoothing over 3 points and 5 ppm mass tolerance and selected MS/MS spectra for the sodium adduct are shown.

MS/MS spectra for all identified species in positive ion mode (raw data).
For the different m/z, the EIC extracted with boxcar smoothing over 3 points and 5 ppm mass tolerance and selected MS/MS spectra for the sodium adduct are shown.
characterization of the identified species from pyruvate cultivation of A. borkumensis based on MS/MS.Characterization was performed for the individual retention times of the double peaks, where MS/MS could be recorded.The fragments detected in positive ion mode are listed, and the conclusions for the composition and position of the fatty acyl characterization of the identified species from hexadecane cultivation of A. borkumensis based on MS/MS.Characterization was performed for the individual retention times of the double peaks, where MS/MS could be recorded.The fragments detected in positive ion mode are listed, and the conclusions for the composition and position of the fatty acyl

Fig. S2
Fig. S2 Fragmentation of the postulated species Glc-40:0-Ala eluting at a retention time of 13.82 min in negative ion mode.MS/MS spectrum for the precursor m/z 930.6 ([M-H] -) acquired with HCD at NCE 18 in the pyruvate culture extract.For a discussion of the observed fragments, see section 3.2

Fig. S5
Fig. S5 MS/MS of Glc-34:0-Gly (m/z 832.5, [M-H] -) in negative ion mode at 3.76 min and 4.05 min at NCE 18 together with the EIC.Measurement of the pyruvate culture extract

Fig. S10
Fig. S10 MS/MS of Glc-40:0-Ala (m/z 930.6, [M-H]-) in negative ion mode at 13.82 min at NCE 18 together with the EIC.Measurement of the pyruvate culture extract

Fig. S32
Fig. S32 MS/MS of 40:0-Gly (m/z 778.5, [M+Na] + ) in positive ion mode at 22.97 min at NCE 30 together with the EIC.Measurement of the pyruvate culture extract

Fig. S34
Fig. S34 Calibration curve for external calibration with 1-monoolein in the 0.8 to 400 mg/L concentration range (equivalent to 3.9 to 2000 ng on the column).A second-order polynomial fit is shown.Measurements were carried out in duplicate; the error bars indicate the range of the results obtained.