A Screening Platform of Alternative Feedstocks for Sophorolipid Production by Starmerella bombicola: Experimental Design, Analytical Methods, and Practices.

Abstract

Increasing scrutiny of the negative ecological and environmental effects of chemical surfactants is driving research into the production and use of biosurfactants that offer low toxicity and high biodegradability. One particular form, sophorolipids, are a strong contender owing to their good surface activity and high production by Starmerella bombicola. Currently, market entry is hindered by high costs linked to the use of first generation ‘high-grade’ feedstocks including glucose and rapeseed oil, that also have significant carbon footprints associated with their use. Explorations into low-cost, eco-friendly alternative feedstocks are underway, but the current approach has by and large been unsuccessful, due in part to the limited approach currently used to screen feedstocks, where feedstocks are tested under generic conditions without consideration of their specific composition. Similarly, analytical methods used to identify and quantify sophorolipid variants vary greatly across the literature and are poorly documented and understood, with no clear comparison of their effectiveness. The work presented in this PhD thesis centres on the development of a feedstock screening platform for alternative feedstocks with Starmerella bombicola, focusing on a fair screening process with a comparison of the most suitable analytical methods. Firstly, a predictive tool that defines media conditions for a feedstock based on its composition was constructed. Secondly, appropriate methods for sample preparation, identification and quantification are explored and developed. The thesis is presented in a Journal (Alternative) Format and is organized into four main research articles. To identify suitable conditions for sophorolipid production, a central composite design of experiments was applied to alter the concentrations of glucose, rapeseed oil and nitrogen (cornsteep liquor/ammonium sulfate). Maximal production of sophorolipids was achieved at 39.5 g/L, improving on the 19.3g/L gained in a commonly cited literature composition. Glucose was not found to have a significant effect on sophorolipid production, which was linked to the production and subsequent consumption of glycerol via rapeseed oil breakdown during the fermentation. Inversely, nitrogen was found to be largely inhibitive to the production process, as increased levels diverted S. bombicola to growth of biomass over sophorolipid production. The work significantly improved the understanding of how S. bombicola utilises feedstocks in the process of sophorolipid production and novelly describes the role of nitrogen and glycerol in the production process. The subsequent predictive model created from the design of experiments results was applied to predict appropriate conditions for wood-derived sugar solutions provided by an industrial manufacturer, determining optimal conditions for production alongside a high-grade feed internal control for comparison. The potential of a C6 sugar rich feed was noted, with 44.3g/L final sophorolipid production, demonstrating a significant improvement compared to the use of pure glucose (29.00 g/L sophorolipid) at identical media concentrations. Investigations into the potential composition of the C6 sugar feeds found acetic acid consumption linked to the improved production levels, which has not been previously reported. Feedstock preparation methods were compared, with tangential flow filtration (TFF) and autoclaving, with TFF allowing an otherwise un-productive feedstock to achieve 40.15g/L sophorolipids, marking a novel method of preparation that can significantly improve the potential of feedstocks. To ensure accurate quantification of the sophorolipids produced by alternative feedstocks, three common analytical methods (anthrone assay, liquid:liquid extraction/gravimetric and high performance liquid chromatography) were compared on 75 unique fermentation broth samples with varying compositions of glucose, rapeseed oil and nitrogen. The HPLC wa

Date of Award	1 Aug 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Peter Martin (Supervisor) & James Winterburn (Supervisor)