Comparative Evaluation of Sophorolipid Production from Olive Mill Wastewater and Palm Oil Mill Effluent

Introduction

The production of sophorolipid biosurfactants has attracted increasing industrial interest owing to their exceptional surface-active properties, biodegradability, and broad-spectrum antimicrobial activity, yet production costs remain a primary barrier to widespread commercialization [1] . Sophorolipids are glycolipid biosurfactants composed of a sophorose disaccharide linked to a hydroxylated fatty acid chain, produced predominantly by the yeast Starmerella bombicola (formerly Candida bombicola) and related species through a unique biosynthetic pathway that incorporates both hydrophilic and hydrophobic carbon substrates [2] . The dual-substrate requirement, typically comprising a sugar and a lipid source, creates a distinctive opportunity for the concurrent utilization of two different categories of agroindustrial waste materials as fermentation feedstocks [3] . Olive mill wastewater and palm oil mill effluent represent two of the most voluminous and environmentally problematic lipid-rich waste streams generated by the global vegetable oil processing industry [4] . Olive mill wastewater, produced at approximately 30 million cubic meters annually across Mediterranean olive-producing regions, contains residual olive oil (0.5-5% w/v), polyphenols, organic acids, and suspended solids that render it highly polluting if discharged untreated [5] . Palm oil mill effluent, generated at a ratio of approximately 2.5 tonnes per tonne of crude palm oil produced, similarly contains residual lipids (4-6 g/L), high biochemical oxygen demand, and recalcitrant organic compounds [6] . The conversion of these problematic waste streams into valuable sophorolipid biosurfactants represents an attractive waste valorization strategy with dual environmental benefits [7] . This research comparatively evaluated the sophorolipid production capacity of Starmerella bombicola KCTC 7882 utilizing olive mill wastewater and palm oil mill effluent as hydrophobic co-substrates under controlled bioreactor fermentation conditions.

Methods

The waste substrates were characterized for total lipid content, chemical oxygen demand, total suspended solids, pH, total phenolic content, and fatty acid composition by gas chromatography-mass spectrometry. Olive mill wastewater was pretreated by centrifugation at 8,000 × g for 15 minutes followed by filtration through Whatman No Figure 1 presents the comparative kinetic profiles of sophorolipid production from both agro-industrial waste substrates. Olive mill wastewater consistently supported higher sophorolipid yields throughout the fermentation period, with the differential becoming increasingly pronounced after 72 hours. The maximum yield of 29.1 g/L was achieved with olive mill wastewater at 168 hours, compared with 24.6 g/L from palm oil mill effluent, representing an 18.3% superiority of the olive-derived substrate. Characterization of the waste substrates revealed distinct compositional profiles that influenced sophorolipid biosynthesis. Olive mill wastewater contained a total lipid content of 3.8 ± 0.3% (w/v) with oleic acid comprising 72.4% of the fatty acid profile, followed by palmitic acid (12.1%) and linoleic acid (8.7%). Palm oil mill effluent exhibited a lower total lipid content of 2.6 ± 0.2% (w/v) with palmitic acid as the dominant fatty acid (44.3%), followed by oleic acid (38.6%) and linoleic acid (10.2%). The higher oleic acid proportion in olive mill wastewater is particularly significant, as Starmerella bombicola preferentially incorporates C18:1 fatty acids into the sophorolipid hydrophobic tail through its cytochrome P450 monooxygenase system. Table 2 presents the summary statistics confirming the statistically significant superiority of olive mill wastewaterderived sophorolipids across all measured physicochemical properties. The higher lactonic-to-acidic sophorolipid ratio (3.2 vs. 2.1) obtained from olive mill wastewater is industrially significant, as lactonic forms exhibit superior surface tension reduction and antimicrobial activity compared with their acidic counterparts. Figure 2 illustrates the emulsification performance of the produced sophorolipids against different hydrocarbon substrates. The highest emulsification indices were observed against kerosene (median 68%), followed by diesel (60%), crude oil (53%), and motor oil (49%). The variation in emulsification performance across hydrocarbon types reflects the influence of substrate viscosity and molecular weight on the formation and stability of oil-in-water emulsions.

Discussion

This research established that both olive mill wastewater and palm oil mill effluent serve as effective hydrophobic cosubstrates for sophorolipid production by Starmerella bombicola, with olive mill wastewater demonstrating statistically significant superiority across all evaluated parameters. The sophorolipid yield of 29.1 g/L achieved from olive mill wastewater compares favorably with literature values ranging from 12 to 40 g/L reported for various lipidic substrates including waste cooking oil, animal fat, and petroleum-derived hydrocarbons [8] .

The superior performance of olive mill wastewater aligns with established understanding that Starmerella bombicola preferentially utilizes oleic acid (C18:1) as the fatty acid precursor for sophorolipid biosynthesis [9] . The predominance of oleic acid in olive mill wastewater (72.4% of total fatty acids) compared with palm oil mill effluent (38.6%) provides a mechanistic explanation for the differential yields. Previous investigations have similarly demonstrated that oleic acid-rich substrates consistently outperform palmitic acid-rich alternatives for sophorolipid production [10] . However, the yield of 24.6 g/L from palm oil mill effluent, while lower than olive mill wastewater, exceeds several reported values for non-optimized sophorolipid fermentations and warrants consideration given the enormous global availability of this waste stream. The palm oil industry generates an estimated 75 million tonnes of effluent annually, representing a vast untapped resource for biosurfactant manufacturing even at lower volumetric yields [11] . The economic feasibility analysis must therefore consider substrate availability and procurement costs alongside volumetric productivity.

The higher lactonic-to-acidic sophorolipid ratio observed with olive mill wastewater (3.2 vs. 2.1) has practical implications for downstream applications. Lactonic sophorolipids exhibit stronger antimicrobial activity against Gram-positive bacteria and demonstrate superior performance as oil-dispersing agents, whereas acidic forms display better foaming characteristics and solubility in aqueous systems [12] . The ability to modulate the congener ratio through substrate selection provides a mechanism for tailoring sophorolipid properties to specific industrial requirements without genetic modification of the producer organism.

Waste Valorization Potential

The conversion of olive mill wastewater and palm oil mill effluent to sophorolipid biosurfactants achieves simultaneous waste treatment and value creation. The chemical oxygen demand of olive mill wastewater was reduced by 68.4% during fermentation, and palm oil mill effluent COD decreased by 57.2%, indicating substantial organic matter removal concurrent with biosurfactant production. This integrated bioprocessing approach addresses regulatory compliance for effluent discharge while generating revenue from the biosurfactant product, supporting the economic sustainability of vegetable oil processing operations.

Conclusion

The principal contribution of this research is the demonstration that lipid-rich agro-industrial effluents can be effectively converted to sophorolipid biosurfactants with yields comparable to refined substrate fermentations, achieving 29.1 g/L from olive mill wastewater and 24.6 g/L from palm oil mill effluent. The superiority of olive mill wastewater is attributable to its higher oleic acid content, which aligns with the substrate specificity of the sophorolipid biosynthetic pathway in Starmerella bombicola. These findings have significance for the biosurfactant manufacturing sector, demonstrating a viable pathway for reducing production costs while simultaneously addressing the environmental burden of vegetable oil processing wastes. Key open questions remain regarding the long-term operational stability of continuous fermentation processes using variable-composition waste streams, the scalability of the integrated waste treatment-biosurfactant production concept, and the market acceptance of wastederived sophorolipid formulations in pharmaceutical and cosmetic applications.

Acknowledgement

AcknowledgementsThis research was supported by the National Research Foundation of Korea (NRF) through funded by the Ministry of Science and ICT. The authors acknowledge the Bioprocess Engineering Laboratory at Seoul Metropolitan University for provision of bioreactor facilities and analytical instrumentation. P.J.H. designed the experimental framework and supervised the investigation; K.S.Y. performed the fermentation experiments and analytical characterization; C.M.W. conducted the statistical analysis and mass spectrometric characterization.

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