Bioprocessing strategies for the cultivation of oleaginous yeasts on glycerol

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Abstract

Over recent years microbial oil has attracted much attention due to its potential to replace traditional oil sources in the production of biofuels and nutraceuticals. Its advantages arise from its independence of the food supply chain and its ease of production compared to conventional plant oils. Also, as concerns for the environment grow, microbially-synthesized oil emerges as potential competitor for the sustainable production of biodiesel. However, the high cost of its production currently hinders its large scale application. The bottlenecks to industrial microbial oil production are the cost of substrate and cultivation. Current research is focusing on process improvements to make microbial oil more competitive and worthwhile to produce. Several types of microorganisms have been explored so far and waste substrates have been utilised as cheap feedstocks. The overall cost is affected by the fermentation stage, therefore it is imperative to design cultivations with little operating requirements and high yields. Consequently, the present thesis aims to contribute to the field by developing and investigating a simple process for oleaginous yeast cultivation, focusing mainly on enhancing the yields during the bioreactor stage.Oleaginous yeasts were screened for their ability to grow on glycerol and the most promising strain was selected for further research. Then, the necessary conditions for its growth and oil accumulation were defined. Shake-flask cultivations showed that the specific growth rate and glycerol consumption of Rh. glutinis were higher at lower glycerol concentrations (≤40 g/L), while higher C/N elemental ratios enhanced oil content. Experimental data were used to construct an unstructured kinetic model to describe and predict the system’s behaviour. The Monod-based model took into account double substrate growth dependence and substrate inhibition. Following that, bioreactor cultivations extended the range of parameters studied, to include the influence of aeration rate and oxygen supply on cellular growth and microbial oil production. Cultivations at different air flow rates were performed in a 2 L bioreactor and showed that a low aeration rate of 0.5 L/min gave the best glycerol and nitrogen uptake rates, resulting in a concentration of biomass of 5.3 g/L with oil content of 33% under simple batch operation. This was improved by 68% to 16.8 g/L (cellular biomass) with similar oil content (34%) by applying a fed-batch strategy.Finally, different glycerol feeding schemes were evaluated in terms of their effect on oil accumulation. The concept of targeting first a cell proliferation stage, limited by the availability of nitrogen, followed by a lipid accumulation stage, fuelled by glycerol was tested. Continual feeding and pulsed feedings, delivering the same total amount of nitrogen (and glycerol), resulted in similar elevated values of both cellular biomass (~25 g/L) and oil content (~40%). Addition of glycerol at higher rates but giving the same total amount of nitrogen led to a further increase in oil content to 53%, resulting in an overall oil yield of more than 16 g/L (the highest achieved throughout the project). With comparable yields to those reported in the literature but achieved with a much poorer medium, there is every reason to be optimistic that microbial oil production from glycerol could be commercially viable in the future.

Keyword(s)

Aeration rate; Biorefinery; C/N ratio; Fed-batch fermentation; Oil content; Oleaginous yeast; Rhodotorula glutinis; Single Cell Oil

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