Surfactants are surface-active compounds that can reduce surface and interfacial tension between immiscible phases, making their application very important in corrosion inhibitors, detergents, wetting agents, oil recovery enhancers and pharmaceutical and cosmetic formulations [1]. As a result, surfactants represent one of the largest categories of synthetic chemicals manufactured worldwide, with their global market size projected to rise to $69.13 billion by 2032 (Precedence Research, 2023). Thus far, the global demand for surfactants has been mainly met by producing synthetic surfactants from petroleum-based, non-renewable sources. In addition to this, synthetic surfactants are often toxic to the environment and non-biodegradable [3]. Hence, global interests have shifted towards surfactants derived from biological sources, commonly referred to as biosurfactants, as alternatives to synthetic surfactants [4]. This includes microbial biosurfactants, which can be produced from renewable carbon sources by various microorganisms, including different strains of bacteria, fungi, and yeasts [5].
Glycolipids are a specific class of microbial biosurfactants which have gained significant interest due to their particularly high production yield and applicability in the cosmetic and personal care industry [6, 7]. Mannosylerythritol lipids (MELs) are a specific glycolipid biosurfactant which have gained interest due to their excellent moisturizing and reparative activity towards dry and damaged human skin cells [8, 9]. In addition to this, MELs have demonstrated selective cytotoxicity towards human skin cancer cells [10]. These properties make MELs an ideal addition to cosmetic and pharmaceutical formulations aimed at treating skin conditions such as eczema, psoriasis, and certain types of skin cancer [11]. In addition to this, MELs have demonstrated excellent reparative activity towards damaged human hair, making them highly applicable in hair care products [12]These properties have been the main driving force behind interest in developing microbial bioprocesses capable of producing high-purity MELs at an industrial scale.
MELs typically consist of a hydrophilic 4-O‐β‐D‐mannopyranosyl‐D‐erythritol core, attached to a varying number of hydrophobic fatty acid chains. Four main structural variants of MELs have been distinguished based on the acylation patterns of the mannose moiety. MEL‐A represents the di‐acylated form of the compound, while MEL‐B and ‐C are mono-acylated at different positions, respectively. Finally, MEL‐D represents the completely deacylated structure of the compound [7]. MEL-A, the most hydrophobic of the MEL variants, has gained particular interest due to their improved surface-active and self-assembly properties [13–15]. It has been established that certain basidiomycetous yeasts, typically of the genus Pseudozyma, and smut fungi belonging to the genus Ustilago, preferentially produce MEL-A as their main structural variant, as presented in Table 1 [7].
Table 1
Common microbial producers of MEL-A and the carbon source used during production [7]
Microbial source | Carbon source | Hydrophobic sugar moiety | Main fatty acid profile | Main structural variant | References |
P. antarctica T-34 | Soybean oil | 4-O-\(\:\beta\:\)-D-mannopyranosyl-D-erythritol | C8:0, C10:0 and C10:1 | MEL-A and MEL-B | [16] |
P. aphidis DSM 70725 or DSM 14930 | Soybean oil | \(\:4-\text{O}-\beta\:-\text{D}-\text{m}\text{a}\text{n}\text{n}\text{o}\text{p}\text{y}\text{r}\text{a}\text{n}\text{o}\text{s}\text{y}\text{l}-\left(1\to\:4\right)-\text{D}-\text{m}\text{e}\text{s}\text{o}-\text{e}\text{r}\text{y}\text{t}\text{h}\text{r}\text{i}\text{t}\text{o}\text{l}\) | C8:0, C10:0 and C10:1 | MEL-A | [17, 18] |
P. crassa CBS 9959 | Oliec acid and glucose | 4-O-\(\:\beta\:\)-D-mannopyranosyl-(2S,3R)-erythritol | C14:0, C14:1, C16:0, C16:1 and C18:1 | Diastereomers of MEL-A, -B and -C | [19] |
P. fusiformata JCM 3931T | Soybean oil | \(\:4-\text{O}-\beta\:-\text{D}-\text{m}\text{a}\text{n}\text{n}\text{o}\text{p}\text{y}\text{r}\text{a}\text{n}\text{o}\text{s}\text{y}\text{l}-\left(1\to\:4\right)-\text{D}-\text{m}\text{e}\text{s}\text{o}-\text{e}\text{r}\text{y}\text{t}\text{h}\text{r}\text{i}\text{t}\text{o}\text{l}\) | C6 and C8 | MEL-A | [20] |
P. rugulosa NBRC 10877 | Soybean oil | \(\:4-\text{O}-\beta\:-\text{D}-\text{m}\text{a}\text{n}\text{n}\text{o}\text{p}\text{y}\text{r}\text{a}\text{n}\text{o}\text{s}\text{y}\text{l}-\left(1\to\:4\right)-\text{D}-\text{m}\text{e}\text{s}\text{o}-\text{e}\text{r}\text{y}\text{t}\text{h}\text{r}\text{i}\text{t}\text{o}\text{l}\) | C8:0, C10:0, C10:1, C16:0, C18:0 and C18:1 | MEL-A | [21] |
U. maydis DSM 4500 or ATCC 1482 | Sunflower oil | 4-O-\(\:\beta\:\)-D-mannopyranosyl-D-erythritol | C6:0, C14:1 and C16:1 | MEL-A | [22] |
Candida sp. SY-16 | Soybean oil | \(\:6-\text{O}-\beta\:-\text{D}-\text{m}\text{a}\text{n}\text{n}\text{o}\text{p}\text{y}\text{r}\text{a}\text{n}\text{o}\text{s}\text{y}\text{l}-\left(1\to\:4\right)-\text{D}-\text{m}\text{e}\text{s}\text{o}-\text{e}\text{r}\text{y}\text{t}\text{h}\text{r}\text{i}\text{t}\text{o}\text{l}\) | C6:0, C12:0, C14:0 and C14:1 | MEL-A | [23] |
Until now, the production of MEL-A has only been considered from hydrophobic carbon sources, such as soybean oil and sunflower oil [7]. However, various challenges arise when using vegetable oils as the carbon source for the production of MEL-A. Firstly, vegetable oils are a relatively expensive substrate, which negatively affects the economic viability of MEL-A production [24]. On the other hand, due to the structural similarity between MELs and residual oils and fatty acids, separation processes, such as chromatography, need to be employed to produce a product with the required purity for cosmetic and pharmaceutical applications. These processes are often challenging to scale and further increase production costs [25, 26]. Consequently, more accessible and cost-effective carbon sources for the production of MEL-A need to be identified.
Ustilago maydis is a corn smut fungus which has shown the ability to produce mainly MEL-A from sunflower oil [22]. However, this organism is closely related to Sporisorium scitamineum (formerly Ustilago scitaminea), a sugarcane smut fungus which has been shown to produce MELs exclusively from hydrophilic carbon sources, such as glucose, fructose, and sucrose [26–28]. Therefore, this study investigated the potential of producing MEL-A from hydrophilic carbon sources by U. maydis DSM 4500 with the aim of identifying a potential route towards addressing the challenges associated with MEL-A production from vegetable oils. To achieve this, the growth and production of MEL-A from various common hydrophilic carbon sources by U. maydis DSM 4500 were considered. The carbon sources considered included glucose, fructose, sucrose, and lactose, which were selected based on their abundance in various industrial waste streams originating from the sugar, fruit and dairy processing industries.
After the production of MEL-A from these sources was established, different factors affecting the growth and MEL-A production by U. maydis were investigated. Despite the importance of a suitable nitrogen source for the growth of U. maydis, it has been established that the expression of certain genes which are essential for the production of MELs are enhanced under nitrogen-limiting conditions [29]. Therefore, different carbon-to-nitrogen (C/N) ratios were considered to understand the relationship between nitrogen availability, growth, and MEL-A production. It has been established that certain trace elements can enhance the expression of specific genes involved in the biosynthesis of MELs. Fan et al., (2014) demonstrated that the microbial production of MEL-A by P. aphidis ZJUDM34 could be increased through the addition of Cu2+, Mn2+ and Mg2+, while Yang et al., (2023) observed a significant increase in the production of MELs by P. aphidis DSM 70725 when the cultures were supplemented with either Fe2+ or Fe3+. Therefore, the potential of achieving enhanced production of MEL-A from hydrophilic carbon sources by U. maydis through the addition of these trace elements was explored in this work. In addition to this, it has been observed that pH significantly affects the production of MELs, with certain strains of basidiomycetous yeasts performing better under neutral conditions, while other strains, including the smut fungus Sporisorium scitamineum, performed better under acidic conditions [32, 33]. Therefore, the effect of pH on the production of MEL-A was also investigated, as shown to have an effect on the final product titre.
After the aforementioned factors were investigated, it was observed that a low C/N ratio supported optimal growth, while a high C/N ratio supported the production of MEL-A. This indicated the potential of maximizing the production of MEL-A from hydrophilic carbon sources by implementing a fed-batch production strategy. This system would maximize biomass formation by providing a medium with a low C/N ratio during growth. Then, upon nitrogen depletion, the cultures could be supplemented with additional carbon sources to support the production of MEL-A. Although this approach has not been previously explored, it has been proven to be a viable route towards achieving significantly enhanced product titres from yeasts of the genus Pseudozyma grown on vegetable oils [34].