Advancing the industrial relevance of the oleaginous yeast Metschnikowia pulcherrima

Abstract

The increasing use of terrestrial oils is associated with sustainability issues such as deforestation. Microbial oils, particularly from yeasts, are a promising alternative, provided they can be produced commercially. To this end, robust oleaginous yeasts with high growth rates are required. Metschnikowia pulcherrima is a robust oleaginous yeast with processing advantages such as exhibiting antimicrobial activity, growing at a low pH and in the presence of fermentation inhibitors- however, to date, relatively low growth rates and yeast biomass concentrations have been reported. To facilitate commercialisation of M. pulcherrima oils, the industrial relevance of this promising, but relatively under-explored oleaginous yeast, must be advanced. In this thesis, this was attempted through better understanding the physiology of the yeast under various operational conditions. The focus was on improving the fermentation growth rates, lipid productivity and yeast biomass concentration with the aim to produce a palm oil substitute.

First, the feedstock scope for M. pulcherrima was expanded by investigating the potential of using macroalgae for fermentation. Hydrolysed through microwave technology, this sustainable feedstock was shown to be suitable for M. pulcherrima lipid production, with brown species superior to red and green. The ten native yeast strains used in this study exhibited a variety of characteristics, from occulation over various growth rates to producing different amounts of 2-phenylethanol and lipids up to 2.6gL 1. Particularly, the polysaccharide breakdown capacity of M. pulcherrima was promising.

However, while the proposed process was promising, the productivity would need to be improved many-fold for a viable process, wherefore process development tools were applied. To this end, an evolved strain of this yeast demonstrated osmophilia growing on sugar solutions as concentrated as 40%(w/v) mediated by the secretion of osmolytes glycerol and arabitol, and also produced ethanol of up to 8.7%(v/v). Through the cultivation at high cell densities of up to 122.6gL 1 (dry cell weight) with a lipid concentration of 50.6gL 1, a lipid productivity of 0.18gL 1 h 1 could be achieved in semi-continuous cultivation with maximum lipid production rates nearly doubled from the batch. To achieve these extreme cell densities, semi-continuous and continuous operation were found to be superior to batch and fed-batch. Excitingly, an oil with similar composition to palm oil could be produced. More influential on the fermentation kinetics, however, was the increased addition of micronutrients. The lipid productivity in batch cultivation could be elevated to 0.29gL 1 h 1, with higher rates likely achievable. At the same time, the production of secondary metabolites was reduced, allowing for increased lipid yields of up to 0.17gg 1 glucose over 50% of the theoretical maximum (0.32gg 1). It was shown that the onset of lipid accumulation is independent of nitrogen limitation with this unique yeast, as opposed to the vast majority of oleaginous yeasts.

Finally, the scalability of M. pulcherrima was demonstrated at the pilot scale. Using hydrolysed starch as a feedstock, the yeast achieved lipid yields of up to 0.21gg 1 consumed saccharides. M. pulcherrima performed equally well at the 2L, 70L and 350L scale in batch and semi-continuous operation mode, for up to 62 days cultivation and with the ability to be cultured on oligosaccharide mixtures.

The work presented in this thesis therefore demonstrates the processing methods required for M. pulcherrima to achieve effective productivity on the industrial scale, opening the way to full commercialisation. These processing methodologies have the potential to be extended to other oleaginous yeasts.

Date of Award	19 Feb 2020
Original language	English
Awarding Institution	University of Bath
Supervisor	Chris Chuck (Supervisor) & Daniel Henk (Supervisor)