Sophorolipid production from different lipid precursors observed with LC-MS

130629 / Ahn Chang ha

The structure of SLs change, it depends on fatty acid precursors

Introduction

Figure1. Molecular structures of the SLs synthesized by T.bombicola

Among the most important biosurfactants are the sohorolipids (SLs) that consist of a sophorose moiety linked glycosidically to a hydroxyl fatty acid residue, as shown in Fig.1

SLs are produced as complex mixtures in the fermentation broth, depending on the carbon sources used

There are many ways to show the structure and ratio of the SLs Isolated SLs from the final fermentation product using liquid chromatography and thin-

layer chromatography (TLC) and identified the structure of a major SL using mass spectroscopy (MS), infrared spectroscopy (IR), and nuclear magnetic resonance (NMR)

Separated the SL components with medium-pressure liquid chromatography (MPLC) and thick-layer chromatography and determined their structures by 1H- and 13C-NMR spectroscopy, fast atom bombardment mass spectroscopy (FAB-MS), and GC/MS

Materials & Methods

Organisms and culture conditions Torulopsis bombicola ATCC 22214 The production medium, with N-source

• Glucose• Second C-source : glucose, hexadecane or soybean oil)• Sodium citrate• Yeast extract• MgSO4

• (NH4)2SO4

• KH2PO4

• FeSO4∙7H2O• CaCl2• NaCl

pH : initial pH (6, with 1N KOH), controlled pH(3.5±0.5, with 3N KOH), in the late stage (3.5, with acid)

Second carbon source was added in 4steps at 24-h intervals• (24, 48, 72, 96h) : the first addition was at the late exponential phase, the

others at the stationary phase• The step-wise additions were adopted to prevent the potential accumulation

of inhibitory fatty acids The agitation and aeration used were 500rpm and 0.5vvm, respectively The temperature was controlled at 30.0°C ± 0.2°C

Materials & Methods

Analytical methods Samples (10ml) taken periodically along the fermentation were extracted twice

with equal volume of ethyl acetate and centrifuged at 14,500g for 10min at room temperature

The cell pellet collected was washed and dried for determining the cell (dry-weight) concentration

The aqueous phase was used for analyses of glucose and ammonium concentrations

The organic phase was vacuum-dried at 40°C to remove the ethyl acetate The residue was twice washed with 10-ml hexane to remove the remaining

hexadecane or soybean oil The crude SLs were obtained after vaporizing the residual hexane at 40 under

vacuum The hexane phase collected from the wash was also dried for determining the

remaining hexadecane or soybean oil

Sophorolipid : HPLC (C-18 column, wave length 207nm, mobile phase 8/2(v/v) acetonitrile/H2O, flow rate 0.5ml/min)

Hexadecane, soybean oil and glucose : HPLC (SI column, mobile phase hexane, flow rate 0.5ml/min)

Cells and ammonium : dry cell weight and ammonia-selective electrode

Structure identification and characterization LC-MS system was used to separate and identify the structures of individual SL

components Mobile phase 8/2(v/v) acetonitrile/H2O The SL components separated by HPLC was passed through the mass

spectrometer where they were ionized by electrospray The molecular ions were collected in an ion trap and the mass/charge (m/z)

values detected

Results

Figure2. Profile of SL production in T.bombicola fermentation using soybean oil as the second C-source

Cells grew rapidly : 1.5-2day

pH dropped to 3.5 : after 1day Ammonium consumption Fatty acid generation

The SL overproduction began at the onset of the stationary phase, paralleling the soybean oil consumption

But later (190h-), extending slightly beyond the time when both glucose and soybean oil were depleted in the medium

Results

Figure3. (a) Mass spectrum of SLs production from glucose alone (b) Mass spectrum of SLs produced from hexadecane as the second C-source (c) Mass spectrum of SLs produced from soybean oil as the second C-source

Fig3(a) : A complex mixture of mainly acidic SLs (with the fatty acid moiety of C18:1 and C16:0, m/z=704.8 and 679.3, respectively) was formed in the fermentation using glucose as the sole C-source

Fig3(c) : The SLs produced with soybean oil as the second C-source were also complex, containing both lactonic SLs (C18:0, C18:1, C18:2 and C16:0 with m/z 688.9, 686.9, 684.9 and 661.2 respectively) and acidic SLs (C18:0, C18:1, C18:2 and C16:0 with m/z=707.0, 705.0, 703.0 and 679.3, respectively)

However, a much cleaner mixture with a single dominant MS peak (lactonic C16:0, m/z=661.3) was obtained with hexadecane as the second C-source

The structures of the corresponding main SL components illustrated in Fig.3

Results

Figure4. MS/MS spectrum from fragmentation of SLs with m/z=661.3

An example of the MS-MS spectra from fragmentation is given in Fig4

The molecular ion (M-) had m/z=661.3

m/z=618.5 and 576.8 corresponded to the ready removal of one and two acetyl groups, respectively, indication that the SL was originally diacetylated

A further loss of one glucose from the de-acetylated structure (m/z=576.8) led to the fragment ion at m/z=432.7

The loss of both sugars gave the final fragment ion at m/z=270.5, corresponding to that from hydroxyl hexadecanoic acid

The MS-MS spectrum, together with the structures reported in the literatures, strongly suggested that the molecule is the diacetylated lactonic SL, L-([2’-O-β-D-glucopyranosyl-β-D-glucopyranosyl] oxy)-hexadecanoic acid 1’-4’’-lactone 6’,6’’-diacetate, as shown in Fig.3b

Results

Figure5. HPLC chromatogram of the crude SLs produced in the fermentation using n-hexadecane as the second C-source

The HPLC chromatograms with the identified structures are shown in Fig.5 for the hexadecane system

As shown in Fig.5, the lactonic SLs, especially the acetylated ones, had longer LC retention times because of their higher hydrophobicity

Two major SLs with the same molecular weight of 662 were observed in the chromatograms for the samples from the hexadecane system

The MS/MS fragmentation analysis showed that the two SL isomers had the same fragments but with slightly different intensity ratios

Results

Figure6. HPLC chromatogram of the crude SLs produced in the fermentation using soybean oil as the second C-source

The HPLC chromatograms with the identified structures are shown in Fig.6 for the soybean oil system

The main SLs obtained had MWs of 688/706 and 686/704

The first pair corresponded to the lactonic/acidic SLs having a C18 fatty acid with one double bond (C18:1), the second pair corresponded to those having a C18 fatty acid with two double bonds (C18:2)

Results

Figure7. Composition change of SLs produced using hexadecane

Figure8. Composition change of SLs produced in the fermentation using soybean oil as the second C-source

The production profiles of individual SL components with hexadecane and soybean oil as the second C-source are shown in Fig.7 and 8

The concentrations of acidic SLs increased with time but at very slow rates

The production of lactonic SLs became appreciable following the addition of hexadecame or soybean oil (at 24h), and increased much more rapidly after the culture reached the stationary phase (at~40h)

Results

Figure9. Change of the percentages of total and major lactonic SLs in the SL mixtures produced in the fermentation with hexadecane or soybean oil as the second C-source

The combined percentage of the three predominant SLs increased significantly and leveledoff at ~80% in the hexadecane system and ~50% in the soybean oil system, respectively

Conclusions

Sophorolipid production by T.bombicola was strongly affected by the use of different lipidic substrates as the second C-source

The production in the fermentation with glucose as the only substrate was far slower than that in the systems supplemented with hexadecane or soybean oil

The yield of crude SLs were 0.84, 0.20, and 0.03 g/gram of hexadecane, soybean oil, and glucose consumed, respectively, during the SL production phase

The results of SL quantification and structural identification obtained with the LC-MS analyses showed that much cleaner mixtures of SLs were produced in the hexadecane system, containing predominantly two diacetylated lactonic SL isomers with palmitate as the fatty acid moiety

A very close structure correspondence between the SL’s lipid moiety and the lipid precursor used in the fermentation was also observed, suggessting that the lipid precursor was converted to the corresponding hydroxyl fatty acid and then incorporated into the SLs

While the concentrations of acidic SLs increased very gradually along the batch fermentation, the production of lactonic SLs became appreciable following the addition of hexadecane or soybean oil at 24h, and increased much more rapidly after the culture reached the stationary phase