Rhamnolipid (RL) is a glycolipid biosurfactant containing rhamnose and β-hydroxylated fatty acid chains with diverse structures and remarkable surface-active properties (Abdel-Mawgoud et al., 2014, Chong and Li, 2017, Dini et al., 2024, Khademolhosseini et al., 2019). RLs have gained recognition for their unique properties and are adaptable to various industrial applications, including the petroleum, food, agriculture, and bioremediation industries (Chebbi et al., 2021, Elshikh et al., 2017).

RLs with longer fatty acid chains exhibit lower critical micelle concentration (CMC) values and better interfacial tension reduction capabilities due to their stronger hydrophobic interactions (Thakur et al., 2021, Zhao et al., 2020). Conversely, RLs with shorter fatty acid chains, typically those with higher E24 values, exhibited better emulsification properties (Zhao et al., 2020).

Pseudomonas aeruginosa has been a well-established RL producer since 1946 (Abdel-Mawgoud et al., 2014, Dobler et al., 2016) but it is also an opportunistic pathogen. This has led to research on safer strains and more cost-effective production methods. Burkholderia thailandensis E264 is a promising alternative for RL production because of its diminished pathogenicity and is classified as a non-pathogenic organism at biosafety level 1 (BSL-1) (Abdel-Mawgoud et al., 2010, Elshikh et al., 2017, Funston et al., 2016).

B. thailandensis E264 has proven its ability to produce RLs as confirmed by the presence of rhlA, rhlB, and rhlC gene orthologues responsible for RL biosynthesis (Dubeau et al., 2009). It predominantly produces di-RLs (Dubeau et al., 2009, Funston et al., 2016, Irorere et al., 2018). The carbon sources available to B. thailandensis E264 can affect RL production, thereby affecting the congener profiles, physicochemical properties, and overall yield (Ji et al., 2016).

Hydrophobic carbon sources, such as vegetable oils, often lead to higher RL yields and better emulsifying properties than hydrophilic sources such as glucose (Chong and Li, 2017, Rocha et al., 2020). However, the use of hydrophobic substrates can increase the oxygen demand, potentially leading to lower growth and RL production (Zhao et al., 2020). Glycerol is a more efficient carbon source for RL production than glucose or other sugars (Mat’átková et al., 2022) . Oleic acid, a C18 fatty acid, significantly improves RL yields compared to other fatty acids (Zhang et al., 2019).

This study employed an untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomic approach to determine the effects of glycerol and oleic acid on RL congener production in B. thailandensis E264. This method allows the annotation of a wide range of RL congeners with different adduct ions, including those not previously reported. The key advantage of this approach is its ability to detect and identify unknown metabolites (Han et al., 2023). This enabled the detection of unexpected RL variations resulting from glycerol and oleic acid as carbon sources.

The primary focus of this study was on secondary metabolite regulation, specifically the regulation of RL production, by focusing on the end-product (RLs) and their changes in response to different carbon sources. Univariate and multivariate analyses were subsequently performed to identify statistically significant RLs responsible for the discrimination pattern in response to different carbon sources. The implications of glycerol and oleic acid as carbon sources for RL production in B. thailandensis were evaluated.