Bioprospecting for a Wild Strain of Sporisorium scitamineum for the Valorization of Sugarcane Molasses into Mannosylerythritol Lipids and Cellobiose Lipids

Abstract

Significant wastes such as bagasse, molasses, and vinasses are produced during sugarcane processing. Due to their high sugar content, these wastes are commonly used as low-cost substrates for biofuel production. However, these substrates are also suitable for the microbial synthesis of high-value biochemicals like biosurfactants. Sporisorium scitamineum, a smut fungus capable of growing on sugarcane residues and producing mannosylerythritol lipids (MELs) and cellobiose lipids (CBLs), was identified as a promising candidate for valorizing sugarcane wastes. This study investigated MEL and CBL co-production from pure sugars and sugarcane molasses using an S. scitamineum strain isolated from sugarcane residues originating from KwaZulu-Natal, South Africa. Among the sugars tested, sucrose supported the highest glycolipid production, yielding 0.24 g/L MELs and 2.73 g/L CBLs. Lower titers were achieved with fructose, and no production occurred with glucose. Sugarcane molasses also proved to be an effective substrate, yielding 1.46 g/L CBLs—the highest reported titer from an industrial waste to date. However, all titers remained far below those of other glycolipids, which consistently exceed 50 g/L. Future efforts should focus on enhancing CBL production through process optimization or genetic engineering.

1. Introduction

Sugarcane cultivation plays a pivotal role in global agriculture, supporting the production of a wide array of products, ranging from foods to fuel. The industry was originally established for the production of sucrose, a natural sweetener that plays a crucial role in the food and beverage industry. However, despite the maturity of sugarcane processing technologies, various by-products are produced during the production of sucrose, including bagasse, molasses, and vinasses [1]. These by-products are typically rich in cellulosic material and residual sugars, making them highly applicable in the paper industry or as feedstocks for the fermentative production of biofuels, such as ethanol, and organic acids [2,3]. However, their composition also makes these by-products potential feedstocks for the microbial production of more valuable bioproducts, such as biosurfactants and other compounds, which can be applied in the agricultural, food, pharmaceutical, and cosmetic industries [1,4,5,6]. Therefore, by identifying microbial strains with the required metabolic capabilities to effectively convert sugarcane wastes into more valuable biochemicals, it could be possible to develop bioprocesses for the microbial valorization of sugarcane wastes into high-value biochemicals.

Sporisorium scitamineum is a phytopathogenic biotrophic fungus responsible for sugarcane smut disease, one of the major diseases affecting sugarcane crops worldwide [7,8]. The disease caused by this fungus was first noted in the Natal region of South Africa in 1877 and has since spread to most of the global regions where sugarcane is cultivated [8]. Sugarcane smut can lead to losses ranging from 30% to total crop loss, and the disease has threatened entire cultivars of sugarcane [8]. As a result, S. scitamineum has been widely studied with the aim of mitigating the threats that this disease poses to the global sugar industry. These studies have led to an improved understanding of the strain’s metabolic capabilities, and, as a result, it has been established that it has the ability to produce different types of glycolipid biosurfactants [9,10,11,12]. Therefore, this strain has been identified as an ideal candidate for the valorization of the different wastes produced during sugarcane processing into high-value glycolipid biosurfactants.

The first of these glycolipids, known as mannosylerythritol lipids (MELs), have gained significant interest due to their excellent moisturizing and reparative activity towards human hair and skin, making them highly applicable in cosmetic products [13]. In addition to this, MELs possess remarkable antibacterial activity towards various strains of Gram-positive bacteria, including various strains of food-borne pathogens [14]. This makes them highly applicable as disinfectants or preservatives in the food industry. On the other hand, interest in the second of these glycolipids, known as cellobiose lipids (CBLs), stems from their antifungal activity towards different strains of animal and plant pathogenic fungi. This property makes them highly applicable in the pharmaceutical and agriculture industries, where they can be used to develop novel antifungal products [15,16,17]. CBLs also possess interesting cosmetic properties, and their gelling characteristics make them ideal for the production of ointments and creams [17,18,19]. In addition to these novel applications, both MELs and CBLs have the potential to replace synthetic surfactants in a wide range of industries [20]. Synthetic surfactants are typically produced from non-renewable petroleum-based sources and are associated with a range of environmental issues [21,22]. Therefore, by replacing synthetic surfactants with biosurfactants produced from sugarcane wastes, significant strides can be made towards a future bio-based circular economy.

As mentioned before, the individual production of both MELs and CBLs from hydrophilic carbon sources by S. scitamineum has been demonstrated. Morita et al. (2009) observed that S. scitamineum NBRC 32730 could produce MELs at a concentration of 6.4 g/L exclusively from sucrose [10]. These results were the first demonstration of the strain’s ability to produce MELs in the absence of hydrophobic substrates, something which is unique to smut fungi of the family Ustilaginaceae [23]. In another study, while investigating the production of CBLs by S. scitamineum DSM 11941, Oraby et al. (2020) reported a product titer in the range of 8.3 g/L when sucrose was used as the sole carbon source [9]. These studies demonstrate the potential of achieving competitive glycolipid titers by growing S. scitamineum exclusively on hydrophilic carbon sources, such as sucrose or sugarcane wastes.

The co-production of MELs and CBLs from glucose, as well as sugarcane molasses, has been previously demonstrated by Ustilago maydis DSM 4500, another smut fungus belonging to the family Ustilaginaceae. However, since U. maydis naturally grows on maize (Zea mays), S. scitamineum might be better sited towards the valorization of sugarcane molasses into MELs and CBLs, since this strain naturally grows on sugarcane residues and is highly adapted towards the nutrient composition thereof [8,24]. However, until now, the co-production of MELs and CBLs by S. scitamineum has not been investigated. Therefore, this paper aimed understand the potential of utilizing sugarcane molasses as a substrate for the co-production of MELs and CBLs by a wild strain of S. scitamineum, isolated directly from infected sugarcane stalks originating from KwaZulu Natal, South Africa, as opposed to U. maydis DSM 4500.

2. Materials and Methods

2.1. Microorganisms

U. maydis DSM 4500 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). A wild strain of S. scitamineum was isolated from infected sugarcane samples provided by the South African Sugarcane Research Institute (SASRI).

2.2. Chemicals

The following chemicals were obtained from Sigma-Aldrich, South Africa: bacteriological agar (for microbiology), D-(−)-fructose (BioReagent, purity 99%), D-(+)-glucose (BioReagent, purity 99%), D-(+)-sucrose (BioReagent, purity 99%), malt extract (for microbiology), magnesium sulfate (purity 98%), monobasic potassium phosphate (purity 99%), mycological peptone, calcium chloride hexahydrate (purity 98%), urea (purity 99%), sodium nitrate (purity 99%), sodium chloride (purity 99%), yeast extract (for microbiology), and p-anisaldehyde (98%). The following solvents were obtained from Sigma-Aldrich, Johannesburg, South Africa: ethyl acetate (purity 99.5%), chloroform (purity 99.8%), methanol (purity 99.9%), ethanol (purity 99%), and ammonium hydroxide (ACS reagent, 28.0–30.0% NH₃ basis). The following acids were obtained from Sigma-Aldrich, South Africa: acetic acid (glacial, 100%) and hydrochloric acid (purity 37%). The following analytical standard was obtained from Cayman Chemical, Ann Arbor, MI, USA: MEL-A (mixture of congeners, ≥95%). Sugarcane molasses was obtained from a local convenience store. The molasses had an approximate sugar composition of 0.179 g sucrose/g molasses, 0.048 g glucose/g molasses, and 0.047 g fructose/g molasses.

2.3. Isolation and Genetic Analysis of Wild-Type S. scitamineum

2.3.1. Isolation of S. scitamineum

Sporisorium scitamineum was isolated from sugarcane samples provided by the South African Sugarcane Research Institute (SASRI). Spores from the samples were suspended in a sterilized 0.1% Tween-80 solution before being dispersed onto Sabouraud dextrose agar (SDA), consisting of 40 g/L dextrose, 10 g/L peptone, and 15 g/L agar, with a pH of around 5.6, and incubated at 30 °C in the dark until growth was observed. Selected growth spots were then sub-cultured onto malt extract agar (MEA) plates until pure cultures were obtained.

2.3.2. DNA Extraction, PCR Amplification, and Sequencing

The full genomic DNA was extracted from the dried sample using a ZR Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA). A total of 10 mg of fungal biomass was used for extraction, which was carried out according to the manufacturer’s instructions. Successful DNA extraction was visualized on a 1% agarose gel with ethidium bromide. The DNA was then stored at −20 °C. Polymerase chain reaction (PCR) amplification was performed on the extracted DNA. Fungal primers were used to target the ITS region of the extracted genome. ITS1 (forward) and ITS4 (reverse) primers were used [25]. Each 10 μL amplification reaction consisted of 4.1 μL Milli-Q water, 5 μL KAPA Taq ReadyMix 2× (Sigma-Aldrich, South Africa), 0.5 μL extracted DNA, and 0.2 μL forward and reverse primers (0.2 mM). A 2720 Thermal Cycler (Thermo Fisher Scientific, Johannesburg, South Africa) was used to carry out the PCR runs with the following parameters: initial denaturing at 94 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 56 °C for 30 s, and a final extension at 72 °C for 7 min, followed by a 4 °C hold. Successful PCRs were visualized using a 1% agarose gel with ethidium bromide. Each 10 μL sequence reaction consisted of 1 μL amplified DNA, 1.25 μL Terminator Buffer, 1 μL BigDye, 1 μL forward primer, and 5.75 μL Milli-Q water. Sequences were obtained at the Central Analytical Facility, Stellenbosch University, using an ABI3730xl with a 50 cm capillary array and POP7.

2.3.3. Phylogenetic Analysis

The sequences generated were visualized and ambiguous bases at the start and end of each sequence were trimmed using Chromas 2.6.6 (https://technelysium.com.au/wp/), after which they were deposited at GenBank (National Center for Biotechnology Information’s database). The generated sequences were run on GenBank’s database using the nucleotide BLAST algorithm. The ITS regions of similar Sporisorium species and an outgroup, Macalpinomyces simplex, were downloaded in the FASTA format [26]. Multiple alignments were performed on the sequences using the MUSCLE Alignment function in Geneious Prime 2021.2.2 [27]. The ends of the aligned sequences were trimmed, and uninformative regions were removed. The ITS alignment was used to perform Bayesian and maximum likelihood analyses. MrBayes 3.2.6 using Geneious Prime was used for Bayesian analysis [28]. The analysis was performed using the HKY85 substitution model. The analysis consisted of four parallel runs of 5,000,000 generations with the sampling frequency set to every 1000 generations, and the posterior probability values calculated after the first 25% of trees were discarded. Further, a maximum likelihood analysis was run using RAxML on Geneious Prime. A total of 1000 rapid bootstrap replicates and the GTRGAMMA model were applied. The best-scoring tree from all the searches was kept [29]. The Bayesian analysis tree is shown with posterior probability values greater than 0.9, and bootstrap values with greater than 75% overlap.

2.4. Culture Conditions

2.4.1. Stock Cultures

Stock cultures were cultivated on an agar medium consisting of malt extract (30 g/L), mycological peptone (5 g/L), and agar (15 g/L) [30]. The cultures were incubated at 30 °C for 4 days before being stored at 4 °C. Stock cultures were renewed every 2 weeks.

2.4.2. Seed Cultures

The seed cultures were prepared in a 50 mL seed medium in 250 mL baffled shake flasks. The medium consisted of glucose (40 g/L), NaNO₃ (3 g/L), yeast extract (1 g/L), KH₂PO₄ (0.3 g/L), and MgSO₄ (0.3 g/L) [10]. The flasks and mediums were autoclaved at 121 °C for 15 min to ensure sterility. The seed cultures were incubated on a rotary shaker (150 rpm) at 30 °C for 3 days.

2.4.3. Co-Production of MELs and CBLs

The co-production of MELs and CBLs from pure hydrophilic carbon sources was investigated by inoculating 100 mL of a glycolipid production medium (GPM) in 500 mL baffled shake flasks with 1 mL seed cultures. The GPM consisted of urea (0.6 g/L), MgSO₄ (0.5 g/L), KH₂PO₄ (1 g/L), NaCl (0.1 g/L), and CaCl₂ (0.1 g/L) [9]. The GPM was individually supplemented with either sucrose (50 g/L), glucose (50 g/L), or fructose (50 g/L) as a carbon source. The pH of the cultures was adjusted to 2.6 by the addition of a 1.0 M HCl solution and was controlled by the addition of a phosphate–citrate buffer, which consisted of citric acid (1.71 g/L) and Na₂HPO₄ (0.31 g/L) [31]. The cultures were incubated on a rotary shaker (150 rpm) at 30 °C for 7 days.

The co-production of MELs and CBLs from sugarcane molasses was investigated by inoculating 100 mL of a sugarcane molasses medium (SMM) in 500 mL baffled shake flasks with 1 mL seed cultures. The SMM consisted of varying concentrations of sugarcane molasses (36 g/L, 72 g/L, and 108 g/L), urea (0.6 g/L), MgSO₄ (0.5 g/L), KH₂PO₄ (1 g/L), NaCl (0.1 g/L), and CaCl₂ (0.1 g/L) [9,32]. The initial pH of the medium was adjusted to 2.6 by the addition of a 1.0 M HCl solution and controlled by the addition of a phosphate–citrate buffer, which consisted of citric acid (1.71 g/L) and Na₂HPO₄ (0.31 g/L) [31].

2.5. Isolation and Purification of MELs and CBLs

To purify the produced CBLs, an extraction procedure described by Oraby et al. (2020) [9] was implemented. A pellet containing biomass and CBL crystals was obtained by centrifuging the cultures at 4400 rpm for 5 min. The resulting pellet was resuspended in acidic water (pH 2.0), which was prepared by decreasing the pH of deionized water with a 10 M HCL solution to remove residual sugars. The biomass and CBLs were collected by centrifugation at 4400 rpm for 5 min. The pellet was resuspended in ethanol at an equal volume to the original culture sample to dissolve the CBLs. The biomass was removed by centrifugation, again at 4400 rpm for 5 min. The ethanol was evaporated under a vacuum to yield a crude CBL extract. To separate the residual MELs, the extract was resuspended twice in ethyl acetate (4 mL per gram). Purified CBLs were collected by centrifuging the suspension at 4400 rpm for 10 min. The ethyl acetate fraction was evaporated under vacuum to yield a MEL-rich extract [9].

TLC purification was employed to obtain highly purified MEL and CBL extracts. The crude glycolipid extracts were dissolved in ethanol at a concentration of 50 g/L. The solutions were then spotted onto TLC plates (Gel 60 F254, Merck, Darmstadt, Germany) and developed with a mobile phase of chloroform/methanol/water (65:15:2). The plates were sprayed with an anisaldehyde solution, which was prepared by adding 0.5 mL of anisaldehyde to 10 mL of glacial acetic acid, before adding 85 mL of methanol and 5 mL of concentrated sulfuric acid. The plates were then heated to 110 °C for 5 min and the CBLs and MELs appeared as purple spots. Silica at the same height as the MEL and CBL spots on subsequent TLC plates was scraped off and suspended in methanol. The silica was removed from the methanol solution by centrifuging the suspension at 13,000 rpm for 10 min. Furthermore, the methanol solution was filtered through a 0.22 μm nylon filter to remove residual silica. After the methanol was evaporated under vacuum, the purified MEL and CBL extracts were obtained.

2.6. Analytical Methods

2.6.1. Microscope Imaging

The micromorphology of the collected spores was studied by rehydrating them in 3% KOH and observing and measuring them under an optical microscope (Nikon Eclipse E800, Tokyo, Japan) with a CFI plain Apochromat VC 100× objective. Thirty spores were selected at random, and spores were measured by length x breadth at 1000× magnification. Measurements are rounded off to the nearest 0.5 μm. Liquid cultures were observed through a Zeiss AxioStar Plus binocular microscope at 20× magnification (Jena, Germany). Images were taken through the eyepiece of the microscope.

2.6.2. UV-Spectrometry

The biomass concentration in the liquid cultures was estimated by measuring the optical density (OD) of culture samples at 600 nm using a UV–visible spectrophotometer. The resulting OD values were converted to biomass concentrations using a pre-established calibration curve.

2.6.3. High-Performance Liquid Chromatography

The concentration of the carbon source in the culture medium was determined using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Johannesburg, South Africa) equipped with a refractive index (RI) detector and a Biorad Aminex HPX-87H column (8 × 300 mm, Bio-Rad, Hercules, CA, USA). Analysis was carried out at 65 °C using 5 mM H₂SO₄ as the mobile phase at a flow rate of 0.6 mL/min.

Nitrate concentrations in the medium were measured using a Dionex Aquion ion chromatography system with a suppressed conductivity detector and a Dionex IonPac AS4A-SC column (4.6 × 250 mm). The separation was performed at room temperature with an eluent composed of 1.8 mM sodium carbonate and 1.7 mM sodium bicarbonate at a flow rate of 1 mL/min.

For glycolipid quantification, culture samples were first diluted 1:1 with ethanol to ensure the complete solubilization of MELs and CBLs. The concentrations of these glycolipids were then determined via HPLC using a silica-based column (RPSep PRX-1, 4.6 × 150 mm) and a low-temperature evaporative light scattering detector. A gradient elution program employing varying methanol/chloroform ratios (from 100:0 to 0:100, v/v) was applied at a flow rate of 1 mL/min.

2.7. Calculations

The product yield per biomass coefficient was calculated with the following equation:

$$Y_{p/b}={\displaystyle \frac{C_{product, final}}{C_{biomass, final}}}$$

(1)

The product yield per carbon source coefficient was calculated with the following equation:

$$Y_{p/c}={\displaystyle \frac{C_{product, final}}{{(C}_{carbon source, initial}-C_{carbon source, final})}}$$

(2)

The product yield per nitrogen source coefficient was calculated with the following equation:

$$Y_{p/n}={\displaystyle \frac{C_{product, final}}{{(C}_{nitrogen source, initial}-C_{nitrogen source, final})}}$$

(3)

The productivity was calculated with the following equation:

$$Y_{p/b}={\displaystyle \frac{C_{product, final}}{t_{days}}}$$

(4)

2.8. Statistical Analysis

The error bars presented in the figures represent the standard deviation of triplicate replicates. The significance of the differences between different means was determined using ANOVA, and differences were defined as statistically significant at a 95% confidence level, or where p < 0.05. All of the statistical analyses were performed in Microsoft Excel.

3. Results

3.1. Isolation and Identification of Wild-Type S. scitamineum

A phylogenic analysis of the spores obtained from the infected sugarcane stalks provided by SASRI, presented in Figure 1, showed similarities in the size and color of the spores to those of S. scitamineum previously described in the literature [22]. The spores were then isolated on SDA to obtain a pure fungal colony, as presented in Figure 2. The BLAST results for the ITS sequence derived from the fungal biomass placed the isolate in the genus Sporisorium, with the sequence providing a match of 100% for multiple S. scitamineum strains. After implementing Bayesian and maximum likelihood analyses on the ITS alignment, a phylogenic tree could be constructed, as presented in Figure 3, showing that the isolated strain was closely related to previously described strains of S. scitamineum.

3.2. The Co-Production of MELs and CBLs from Pure Hydrophilic Carbon Sources by Wild-Type S. scitamineum

The isolated strain’s ability to produce glycolipid biosurfactants from pure hydrophilic carbon sources was investigated by growing the strain under conditions which were previously shown to facilitate the production of MELs and CBLs by S. scitamineum DSM 11941 and U. maydis DSM 4500 [9,30]. The S. scitamineum wild strain was grown on the defined GPM, supplemented with sucrose at a concentration of 50 g/L as the carbon source, at a pH of 6.0 and 2.6 to demonstrate the strain’s ability to produce CBLs. As shown in the microscopic images in Figure 4, the formation of needle-like CBL crystals was observed when the pH of the GPM was decreased to pH 2.6. These observations were consistent with those made in previous studies, which investigated the effect of pH on the fermentative production of CBLs in submerged cultures [9,31]. Importantly, these observations qualitatively demonstrate the wild strain’s ability to produce CBLs under suitable conditions. However, the structure of these compounds will be verified by nuclear magnetic resonance (NMR).

The time course of the co-production of MELs and CBLs by the wild strain of S. scitamineum from sucrose, glucose, and fructose is shown in Figure 5. These sugars were specifically selected based on their presence at significant levels in various waste streams originating from the sugarcane processing industry, the same wastes that this study aimed to valorize into glycolipid biosurfactants. It was observed that the organism grew comparatively well on the different sugars, reaching biomass concentrations in the range of 4.30 ± 0.20 g/L, 3.76 ± 0.19 g/L, and 3.05 ± 0.13 g/L on sucrose, glucose, and fructose, respectively, when the stationary growth phase was reached between day 3 and 4. These values demonstrate the strain’s ability to metabolize all of the sugars present in sugarcane molasses. Perhaps unsurprisingly, the highest biomass titer was achieved in the presence of sucrose, which is the most abundant of the three sugars in sugarcane, the strain’s natural host.

Similar biomass titers were reported by Oraby et al. (2020) [9], who achieved a biomass concentration of 4.6 g/L by growing S. scitamineum DSM 11941 on sucrose at a pH of 2.5. Importantly, the strain demonstrated significantly improved biomass titers under these conditions in comparison to U. maydis DSM 4500, another smut fungus with the ability to co-produce MELs and CBLs. It was previously established that both MEL and CBL production by U. maydis DSM 4500 and S. scitamineum DSM 11941 is enhanced under nitrogen-depleted and increasingly acidic conditions [9,29]. It was established that the low-biomass titers achieved under these conditions by U. maydis was one of the main factors inhibiting the production of MELs and CBLs. However, these results demonstrate that S. scitamineum can be more efficiently grown under the conditions suited towards the production of glycolipids in comparison to U. maydis.

As presented in

Table 1. The co-production of MELs and CBLs by a wild strain of S. scitamineum from sucrose, glucose, and fructose at a concentration of 50 g/L.

Carbon Source	CBLs					MELs
	Titre (g/L)	Yield per Biomass Formed (g/g)	Yield per Carbon Consumed (g/g)	Yield per Urea Consumed (g/g)	Production Rate (g/L·d)	Titre (g/L)	Yield per Biomass Formed (g/g)	Yield per Carbon Consumed (g/g)	Yield per Urea Consumed (g/g)	Production Rate (g/L·d)
50 g/L sucrose	2.727 ± 0.680	0.701 ± 0.785	0.075 ± 0.019	4.544 ± 1.133	0.085	0.236 ± 0.046	0.061 ± 0.053	0.007 ± 0.001	0.394 ± 0.077	0.006
50 g/L glucose	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0
50 g/L fructose	0.711 ± 0.615	0.208 ± 1.091	0.064 ± 0.055	1.186 ± 1.025	0.077	0.022 ± 0.033	0.007 ± 0.058	0.002 ± 0.003	0.037 ± 0.054	0.004

The co-production of MELs and CBLs from sugarcane molasses by wild-type S. scitamineum.

, when sucrose was used as the carbon source, final MEL and CBL titers of 0.24 ± 0.05 g/L and 2.73 ± 0.68 g/L were achieved, respectively, after 7 days of fermentation. On the other hand, fructose led to final MEL and CBL titers of 0.02 ± 0.03 g/L and 0.71 ± 0.62 g/L, respectively, and no glycolipid production was observed when glucose was used as the carbon source. After performing a single-factor ANOVA analysis, it was deemed that the effect of the sugar used during the fermentation on the production of both MELs and CBLs was statistically significant (p < 0.05). This observation demonstrates that sucrose was the most suitable hydrophilic carbon source for the co-production of MELs and CBLs by this strain of S. scitamineum. These results are unsurprising since the organism naturally grows on sugarcane residues with high concentrations of sucrose. The suitability of utilizing sucrose as opposed to glucose or fructose for the co-production of MELs and CBLs by this strain was further demonstrated by considering the yield of both CBLs and MELs per biomass in the presence of the different sugars, also presented in Table 1. Utilizing sucrose led to a CBL yield per biomass of 0.701 g CBL/g biomass and a MEL yield per biomass of 0.061 g MEL/g biomass. This was significantly higher than the yields achieved in the presence of both glucose and fructose, demonstrating the potential of utilizing sucrose-rich waste for the co-production of MELs and CBLs by S. scitamineum.

By utilizing fructose, both CBL and MEL production was observed, although at a lower efficiency in comparison to sucrose. On the other hand, although the organism grew effectively in the presence of glucose, no MEL or CBL production was observed in the presence of this carbon source. These results indicated that the strain preferentially converts fructose into glycolipids as opposed to glucose. On the other hand, increased biomass concentrations were achieved in the presence of glucose in comparison to fructose. These observations indicate that the strain preferentially utilized glucose for biomass formation, while fructose was preferred for glycolipid formation. Therefore, although waste streams with high concentrations of glucose are clearly not suitable for the co-production of MELs and CBLs by this strain, they are suited for the growth of the strain. In addition to this, these observations indicate that waste streams with high glucose concentrations could be subject to enzymatic pretreatment processes to convert the glucose into fructose, increasing their suitability as substrates to produce glycolipids by S. scitamineum. This potentially opens the door to the utilization of a whole range of industrial wastes to produce glycolipids by this strain. However, this approach needs to be investigated further.

From both Figure 5 and Table 1, it was observed that under these conditions, the strain produced CBLs more effectively than MELs in the presence of hydrophilic carbon sources. In a previous work, it was established that MEL production by basidiomycetous yeasts and fungi is more dependent on the presence of hydrophobic, lipid-rich substrates in comparison to the production of CBLs [27]. This could be explained by looking at the biosynthetic pathway for both MELs and CBLs by these strains. In the absence of hydrophobic substrates, palmitic acid is synthesized directly from the hydrophilic carbon source as the last common hydrophobic precursor to the fatty acid constituents in both MELs and CBLs [32,33]. To produce the short-chain fatty acids present in MELs, the palmitic acid undergoes $\beta$-oxidation, also known as the chain-shortening pathway [34,35]. Alternatively, to produce the hydrophobic 15,16-dihydroxypalmitic acid moiety that is present in CBLs, the palmitic acid undergoes double hydroxylation, which is facilitated by two monooxygenases [36]. Therefore, it became clear that, under these conditions, the wild strain of S. scitamineum preferentially produced 15,16-dihydroxypalmitic acid as opposed to the short-chain fatty acids that are required for the synthesis of MELs. These findings indicate the potential of implementing more advanced genetic engineering techniques, such as knocking the genes out of either the CBL or MEL biosynthetic pathways, to develop a modified strain that either exclusively produces CBLs at more competitive titers or has the ability to produce MELs from hydrophilic carbon sources more effectively, addressing challenges associated with the downstream processing of MELs contaminated with fatty acids.

Significantly decreased MEL titers were observed in comparison to those reported by Morita et al. (2009) [27], who achieved a MEL titer of 6.4 g/L by growing S. scitamineum NBRC 32730 on a medium consisting of sucrose at a concentration of 100 g/L and NaNO₃ at a concentration of 3 g/L. In addition to this, the pH of the medium was controlled at 6.0 and the fermentation was performed at a temperature of 25 °C [27]. These observations indicate the significant impact of the carbon source concentration, C/N ratio, pH, and temperature on the production of MELs from hydrophilic carbon sources by S. scitamineum. Furthermore, these observations indicate that the production of MELs by this strain was not enhanced under acidic conditions, as was the case for U. maydis DSM 4500 [29]. These observations indicate the need for further research focused on the optimization of operating conditions for the potential efficient production of MELs from hydrophilic carbon sources by this strain.

The ability of the wild strain of S. scitamineum to produce glycolipid biosurfactants, especially CBLs, from the different sugars present in sugarcane was demonstrated in the previous section. These results presented S. scitamineum as a potential future candidate for the valorization of wastes derived from the sugarcane processing industry, such as sugarcane molasses, into high-value glycolipid biosurfactants. In order to further demonstrate this, the co-production of MELs and CBLs by a wild S. scitamineum strain was demonstrated from varying concentrations of sugarcane molasses, as shown in Figure 6. The molasses concentrations used in this study consisted of 0.179 g sucrose/g molasses, 0.048 g glucose/g molasses, and 0.047 g fructose/g molasses. Therefore, the initial sugarcane molasses concentrations of 36 g/L, 72 g/L, and 108 g/L represented total initial sugar concentrations of approximately 10 g/L, 20 g/L, and 40 g/L, respectively.

As shown in Figure 6 and

Table 2. The co-production of MELs and CBLs by a wild strain of S. scitamineum from varying concentrations of sugarcane molasses.

Carbon Source	CBLs				MELs
	Titre (g/L)	Yield per Carbon Consumed (g/g)	Yield per Urea Consumed (g/g)	Production Rate (g/L·d)	Titre (g/L)	Yield per Carbon Consumed (g/g)	Yield per Urea Consumed (g/g)	Production Rate (g/L·d)
36 g/L sugarcane molasses	0.750 ± 0.478	0.138 ± 0.088	1.250 ± 0.797	0.107	0.246 ± 0.160	0.045 ± 0.030	0.411 ± 0.267	0.035
72 g/L sugarcane molasses	1.001 ± 0.106	0.088 ± 0.009	1.668 ± 0.176	0.143	0.339 ± 0.044	0.030 ± 0.004	0.565 ± 0.074	0.048
108 g/L sugarcane molasses	1.463 ± 0.039	0.072 ± 0.002	2.439 ± 0.065	0.209	0.170 ± 0.032	0.008 ± 0.002	0.283 ± 0.054	0.024

, the highest CBL titer of 1.46 ± 0.04 g/L was achieved at a sugarcane molasses concentration of 108 g/L, representing the highest CBL titer achieved from an industrial waste to date. Final CBL titers of 0.75 ± 0.48 g/L and 1.00 ± 0.1 g/L were achieved when sugarcane molasses was supplemented to the medium at a concentration of 36 g/L and 72 g/L, respectively. However, although the CBL titers increased with an increasing sugarcane molasses concentration, an opposite trend was observed in the CBL yield per carbon consumed. A maximum CBL yield per carbon consumed of 0.138 g/g could be achieved at a sugarcane molasses concentration of 36 g/L, representing the highest product yield per carbon consumed achieved in this entire study. The CBL yield per carbon consumed decreased to 0.088 g/g and 0.072 g/g when the medium was supplemented with sugarcane molasses at a concentration of 72 g/L and 108 g/L, respectively. This was likely due to the increased concentration of unconsumed sugar in the medium at the end of the fermentation when the culture was supplemented with sugarcane molasses at higher concentrations, as shown in Figure 6. However, it is important to note that the effect of the concentration of sugarcane molasses on the production of CBLs by wild-type S. scitamineum was deemed statistically insignificant (p > 0.05) after performing a single-factor ANOVA analysis.

The maximum CBL titer of 1.46 ± 0.04 g/L achieved by the wild strain of S. scitamineum represented a statistically significant improvement (p < 0.05) in comparison to the CBL titer of 0. 492 g/L achieved by growing U. maydis DSM 4500 on sugarcane molasses in a previous study. As previously discussed, U. maydis is a smut fungus that is adapted for growth on maize plants. Starch, consisting of either linear or highly branched chains of glucose, is the main substrate available in maize plants [37,38]. Therefore, it is unsurprising that U. maydis was less effective in producing CBLs from sugarcane molasses in comparison to S. scitamineum, which is naturally adapted to grow on sugarcane residues [7].

Sugarcane molasses proved to be a less suitable substrate for the production of MELs in comparison to CBLs, with final MEL titers of 0.246 g/L, 0.339 g/L, and 0.17 g/L being achieved at sugarcane molasses concentrations of 36 g/L, 72 g/L, and 108 g/L, respectively. These results were unsurprising, since it has been previously established that MEL production is more dependent on the presence of a hydrophobic carbon source when compared to CBL production [27,29]. However, similar to the production of CBLs from sugarcane molasses by this strain, the effect of the concentration of sugarcane molasses on the production of CBLs by wild-type S. scitamineum was deemed statistically insignificant (p > 0.05) after performing a single-factor ANOVA analysis. However, by comparing the MEL titers and yields achieved by the wild strain of S. scitamineum grown on sugarcane molasses, presented in Table 2, to those achieved from the same strain grown on pure carbon sources, presented in Table 1, it was evident that the utilization of sugarcane molasses led to significantly increased MEL titers and yields. This was likely due to the significant amount of complex nitrogen sources, as well as a wide range of mineral ions in sugarcane molasses, which likely play a crucial role in the growth and metabolic performance of this strain [6,39]. Similar observations were made by Morita et al. (2009 and 2011), who reported MEL titers of 6.4 g/L and 12.7 g/L by growing S. scitamineum NBRC 32730 on sucrose and sugarcane juice, respectively [6,27]. As mentioned before, the increased MEL titers achieved in these studies were attributed to variations in the C/N ratio, pH, and temperature under which the fermentations were performed. These observations clearly demonstrated the suitability of sugarcane wastes to produce glycolipids by S. scitamineum. However, more work should be focused on understanding how different operating conditions affect the strain’s ability to produce MELs.

4. Conclusions

This work investigated the co-production of MELs and CBLs from sugarcane molasses by a wild S. scitamineum strain isolated from an infected sugarcane field in KwaZulu-Natal, South Africa. The strain was isolated by germinating spores collected from the infected sugarcane stalks on SDA, before isolating pure fungal colonies on MEA. The identity of the strain was verified by performing an in-depth genetic and phylogenic analysis. Thereafter, the isolated strain achieved CBL and MEL titers of 2.73 ± 0.68 g/L and 0.24 ± 0.05 g/L, respectively, when grown on a medium supplemented with sucrose at a concentration of 50 g/L as the carbon source and at a pH of 2.6 and a temperature of 30 °C. In terms of MEL production, the titer was significantly lower than those reported for S. scitamineum NBRC 32730 grown on a medium supplemented with sucrose at a concentration of 100 g/L, a pH of 6.0, and a temperature of 25 °C. These observations indicate that S. scitamineum produces MELs more efficiently under neutral conditions, and that temperature significantly affects the strain’s ability to produce MELs. However, more research should be directed towards understanding how these operating conditions affect glycolipid production by S. scitamineum. It was observed that both fructose and glucose were less-suitable carbon sources for the production of glycolipids by the wild strain of S. scitamineum, with fructose leading to a final CBL and MEL titer of 0.208 g/L and 0.064 g/L, respectively, while no glycolipid production could be achieved from glucose. These results indicate that sucrose-rich wastes, such as by-products from industrial sugar refining, could potentially be used for the production of glycolipids by these strains. Although competitive MEL titers could not be achieved from the sugarcane molasses, the CBL titer of 1.46 g/L represents the highest CBL titer achieved from an industrial waste stream to date. Therefore, this study demonstrates the potential of valorizing sucrose-rich waste streams derived from industrial sugar refining for the production of CBLs. However, more research should be focused on optimizing the operating conditions in order to maximize the conversion of sugarcane wastes into CBLs. Furthermore, more advanced microbiology techniques should be explored in order to understand the biosynthetic pathway for glycolipid synthesis in this strain, as well as to develop genetically enhanced S. scitamineum strains with improved glycolipid production capabilities. Finally, further techno-economic work is required in order to demonstrate the economic viability of producing CBLs from sugarcane wastes in comparison to the utilization of pure substrates.