The development and evaluation of engineering strategies to enhance microbial oil production for industrial applications
Over the last few decades, microbial oils have gained the attention of researchers worldwide for their potential as sustainable and renewable alternatives to current oil sources. The extensive research into these microorganisms has discovered that microbial oil production offers multiple advantages over current commercial oil sources such as the utilisation of various carbon sources for cultivation (including industrial wastes and by-products), high volumetric production and seasonally independent production. Large-scale microbial oil production has been successful for the production of high value oils such as those rich in polyunsaturated fatty acids (PUFAs) however, commercial production of microbial oil remains hindered by the complexity and high operating costs associated with the process. This renders production economically inviable for mid- or low-value products. Given the extensive research performed on oleaginous microorganisms, strain selection can be complicated and overwhelming. Therefore, a pre-optimised objective function was developed to qualitatively analyse disparate literature data to score and select a set of preliminary production strains for consideration. High sensitivity to changes in literature data, as well as the uncertainty associated with it, highlighting the need for a second screening phase whereby selected strains are experimentally compared. Experimental comparison highlighted the need for more descriptive selection criteria in the objective function and was successful in selecting strains with high biomass and lipid production capabilities for large-scale production and further study in this dissertation. Rapid and efficient lipid quantification techniques are required to monitor lipid accumulation and optimise lipid extraction time, given that oleaginous microorganisms consume their accumulated lipids under extracellular carbon limitation. Gravimetric determination of total lipid content using the Folch method is considered a research standard however, it is laborious and time consuming. Therefore, the accuracy and practicality of two alternative lipid quantification methods - flow cytometry and a Sudan Black B based spectrophotometric method - were evaluated against the Folch extraction method. Results reveal that while the spectrophotometric method yielded slightly more reliable results for in situ lipid quantification than flow cytometry, particularly when used with R. toruloides, neither alternative method demonstrated sufficient accuracy to replace gravimetric estimation of total lipid content. Microscope imaging was used to identify the main limitation of the dyes used in these methods, and found that in situ lipid quantification is highly strain specific with cell wall thickness, fatty acid composition and localisation of microbial oils strongly influencing dye uptake and quantification measurements. The systematic evaluation of the effect of temperature and aeration on the growth, lipid accumulation and fatty acid composition of two oleaginous yeast, and two thraustochytrid strains revealed that oleaginous yeasts are more amenable to manipulation by temperature than thraustochytrids. Yeast strains exhibited strong cold adaptive traits, achieving high specific growth rates and lipid productivity, and biomass concentrations of up to 14 g/L when cultivated at 10°C. Thraustochytrid strains remained relatively consistent biomass concentration and yields across all temperatures, with extended lag phases under cold conditions. While cold conditions were found to enhance lipid accumulation as well as substrate conversion efficiency in all strains used, it was found that cultivation at 20°C and 10°C promotes the production of polyunsaturated fatty acids and monounsaturated fatty acids, respectively, in both oleaginous thraustochytrids and yeasts. Air sparging of the experimental cultures was found to significantly reduce the cultivation times of all strains and, in some cases, enhane biomass and lipid production. Aeration was also found to reduce the effects of FA variation with cultivation temperature in Y. lipolytica and 5. limacinum, and reduce the production of C20, C22 and C24 FAs in all strains, indicating that aeration has a pronounced effect on the FAS enzyme, which is associated with the elongation of fatty acids in the lipid synthetic metabolism of oleaginous microorganisms. These findings demonstrate that temperature can be leveraged as a non-genetic strategy to direct lipid yield and composition, thus offering new opportunities for optimising sustainable microbial oil production. Finally, a novel microbial oil production plant employing a two-stage strategy was designed using Yarrowia lipolytica and 5chizochytrium limacinum, with an estimated economic accuracy of approximately 50%. While the process achieved annual lipid outputs of 22,305 L for Y. lipolytica and 5,311 L for 5. limacinum, using cultivation data from Fontanille et al. (2012} and Pawar et al. (2021), economic analysis showed that neither microbial oil is competitive with current edible or cosmetic oils, requiring minimum selling prices (MSPs} of $502-$666/L and $3,072-$4,006/L, respectively, to achieve an internal rate of return of 20%-35%. However, it was found that the minimum selling price of Y. lipolytica oils fall within the range of commercial omega-3/6 supplement prices, thereby highlighting the importance of FA profile in high-value microbial oil applications. Sensitivity analysis on the economic model confirmed that dilution rate, biomass concentration and lipid content are the dominant economic drivers for this process. Overall, this study demonstrates that a two-stage design separating biomass accumulation and lipid production, especially when paired with temperature-shift and low-cost substrates, provides a scalable and economically promising framework for future microbial oil bioprocess optimisation and commercialisation. Through the implementation of novel strategies and designs for large-scale microbial oil production, the work presented in this dissertation establishes an economically viable framework for sustainable large-scale microbial oil production. Through the combination of incorporating engineering strategies and biotechnological techniques, the studies this work comprises of successfully develops and evaluates engineering strategies that can be used to enhance industrial-scale microbial oil production.
Networked Digital Library of Theses and Dissertations