Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene

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Abstract

Liquid chromatography/mass spectrometry using electrospray ionisation was used to analyse rhamnolipids produced by a Pseudomonas aeruginosa strain with mannitol or naphthalene as carbon source. Identification and quantification of 28 different rhamnolipid congeners was accomplished using a reverse-phase C18 column and a 30 min chromatographic run. Isomeric rhamnolipids that were not chromatographically resolved could be identified by interpretation of their mass spectra and their relative proportions estimated. The most abundant rhamnolipid produced on mannitol contained two rhamnoses and two 3-hydroxydecanoic acid groups. The most abundant rhamnolipid produced from naphthalene contained two rhamnoses and one 3-hydroxydecanoic acid group.

Introduction

Biosurfactants are surface-active molecules synthesised by a variety of microorganisms [1]. These compounds are receiving considerable attention as they could find many potential industrial and environmental applications, especially as substitutes for synthetic surfactants [2]. For example, in bioremediation processes, they can promote the biodegradation of hydrophobic pollutants such as hydrocarbons by emulsification and solubilisation [3]. Bacteria of the Pseudomonas genus are known to produce glycolipid-type surfactants containing rhamnose and 3-hydroxy fatty acids [4]. The exact physiological role of rhamnolipids is still undetermined. Besides promoting growth on liquid n-alkanes [5], rhamnolipids are produced among a variety of extracellular virulence factors involved in opportunistic human infections in immunocompromised individuals and people with cystic fibrosis [6], [7]. Many potential applications of rhamnolipids have been described. They have been shown to exhibit antimicrobial activity against competing microorganisms [8], to be effective in biological control of zoosporic phytopathogens [9], and to facilitate the removal of heavy metals from soil [10].

The two main rhamnolipids produced by Pseudomonas aeruginosa in liquid cultures are L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-C10-C10) and L-rhamnosyl-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C10-C10) [11]. According to the biosynthetic pathway proposed by Burger et al. [12], rhamnolipid synthesis proceeds by sequential glycosyl transfer reactions, each catalysed by a different rhamnosyltransferase. The rhl genes encoding the first rhamnosyltransferase, which catalyses the transfer of TDP-L-rhamnose to 3-hydroxydecanoyl-3-hydroxydecanoate, in addition to the regulatory genes involved, have been characterised [13], [14], [15]. Moreover, an NADPH-dependent 3-ketoacyl reductase required for the synthesis of the 3-hydroxy fatty acid moiety of rhamnolipids was recently identified [16].

In recent years, some publications have reported that Rha-C10-C10 and Rha-Rha-C10-C10 are in fact produced as part of a complex mixture of rhamnolipids [17], [18], [19], [20]. These compounds all contain one or two rhamnose groups linked to one or two 3-hydroxy fatty acids of different chain length, which may contain one double bound. The various combinations of these groups generate a large number of possible rhamnolipid congeners. The methods used for the isolation and chemical analysis of rhamnolipids in these few studies all involved an initial chromatographic separation of the mixtures into various fractions by thin-layer chromatography, often followed by high-performance liquid chromatography (HPLC). These semi-purified fractions were then analysed by mass spectrometry, mostly by fast atom bombardment (FAB) ionisation. Although these methods give excellent information on the structure of the different isolated rhamnolipids, they are of little help in the study of the complete profile of the mixtures, as some congeners may be lost throughout the various purification steps.

As part of a project aiming to elucidate the importance of biosurfactant production in the biodegradation of petroleum hydrocarbon pollutants, we have previously reported the isolation of Pseudomonas strains able to produce glycolipidic biosurfactants, presumably rhamnolipids, while growing on the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene [21]. Since growth of these bacteria on PAHs gives only low amounts of biosurfactants and is accompanied by the accumulation of interfering pigments, a new procedure was required to analyse and quantify the glycolipids produced. In the present study, we report a simple and efficient method to characterise the profile of the rhamnolipids secreted by a strain of P. aeruginosa grown on a water-soluble substrate (mannitol) and a poorly water-soluble substrate (naphthalene). This method involves liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/MS). The profile of the various rhamnolipids produced with these different carbon sources will be described.

Section snippets

Microorganism

P. aeruginosa 57RP was isolated from a hydrocarbon-contaminated soil. It is one of the strains previously selected for their ability to produce biosurfactants when growing on the PAHs naphthalene and phenanthrene [21].

Cultivation and rhamnolipid production

Bacteria were grown in 2-l Erlenmeyer flasks containing 500 ml of iron-limited mineral salts medium supplemented with 2% (w/v) mannitol or naphthalene. The composition of the medium was (g/l): KH2PO4, 0.7; Na2HPO4, 0.9; NaNO3, 2.0; MgSO4·7H2O, 0.4; CaCl2·2H2O; and FeSO4·7H2O,

Results

In the mannitol culture, 2.311 g of purified rhamnolipid extract were obtained after 14 days of incubation, whereas about 180 mg of rhamnolipid extract were recovered from the naphthalene culture after 7 days of incubation. Previous experiments had shown that rhamnolipids from naphthalene cultures are very difficult to purify, apparently because relatively low concentrations are produced. Furthermore, an intense brownish red pigment resulting from naphthalene metabolism accumulates in the

Discussion

LC/MS was very efficient for the analysis of complex rhamnolipid mixtures, in a relatively short and simple chromatographic run. Schenk et al. [23] also used HPLC to characterise a rhamnolipid mixture produced by P. aeruginosa strain DSM 2659. Their method required derivatisation with para-bromoacetophenone in order to detect the rhamnolipids with UV. This method, in addition to being longer than the one described here, could not provide structural information on the various compounds

Conclusion

LC/MS using electrospray ionisation is a simple method for studying the rhamnolipid mixture profile synthesised by P. aeruginosa. It was especially useful for the analysis of rhamnolipids produced on naphthalene, since the accumulation in the culture supernatant of coloured naphthalene breakdown products prevented the use of other methods such as thin-layer chromatography. In this procedure, mass spectrometry allowed analysis of isomeric rhamnolipids that were not chromatographically resolved.

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