Cui, Jing; (2022) Evaluation of a low-cost single-use photobioreactor and its application in the cultivation of microalgae for recombinant protein production. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Recent advances in genetic engineering technologies for microalgae are providing new potential for their use as cell factories for the synthesis of recombinant proteins. As phototrophs, their low-cost and simple cultivation using light energy and CO2 as inputs also make microalgae an attractive host for sustainable production. However, the development of efficient upstream bioprocessing is still in its infancy and many challenges exist, such as low biomass yields and low heterologous protein expression. The research described in this thesis focuses on phototrophic cultivation of microalgae and explores the potential of the chloroplast of Chlamydomonas reinhardtii as a sub-cellular compartment for recombinant protein expression. Chlorella sorokiniana and C. reinhardtii were used as model microalgae in this thesis because of their robustness and potential as platform strains for biotechnology applications. The work is conceptualised from three aspects: photobioreactor (PBR) technology, cultivation strategies, and cell engineering. The first study characterises the engineering parameters of a low-cost disposable hanging bag (HB)-type PBR, including the mass transfer coefficient, mixing time, and gas holdup. Several sparger designs were investigated for increasing gas-liquid mass transfer in the HB. Design improvements focusing on superior sparging were deliberately constrained to maintain simplicity and cost-efficiency. A multi-orifice pipe sparger produced from a modified pipette tip was the most effective. In combination with the enlarged surface-to-volume ratio of the bags, the addition of the new sparger to the HB system resulted in a 53% (three-day biomass productivity) increase in phototrophic biomass productivity of C. sorokiniana when compared with the original system. Despite this improvement, the achievable biomass density was low, which may be problematic for downstream dewatering. To address this, a phototrophic fed-batch cultivation strategy was investigated in the HB system. A bolus feeding strategy using a timed schedule with continuous bubbling of CO2-enriched air was developed and applied to C. sorokiniana, C. reinhardtii, and a luciferase-expressing transplastomic C. reinhardtii strain. All strains showed more than a seven-fold increase in culture density compared to representative batch cultures, although the recombinant luciferase protein did not accumulate to proportionally high level in the culture. In addition, two ‘proof-of-concept’ studies were carried out to explore whether new genetic engineering tools could be developed for microalgae that would contribute to improved recombinant protein productivity, either by enhancing photosynthesis or by allowing synthesis of complex recombinant proteins through a protein-splicing process. In the first experiment, a green light-absorbing proteorhodopsin (GPR) was expressed in the C. reinhardtii chloroplast to improve photosynthesis efficiency by utilising the entire visible light spectrum. The GPR-expressing strain showed improved biomass productivities under phototrophic fed-batch conditions. In the second study, a yellow fluorescent protein with split inteins, a protein precursor that induces protein splicing, was designed and expressed in the C. reinhardtii chloroplast. Whilst the split inteins mediated protein ligation and were able to reconstitute the fluorescent protein in the Escherichia coli model, this was not seen in the chloroplast of the C. reinhardtii model. This was hypothesised to be caused by poor folding or solubility of the intein segments in the chloroplast, which could be a focus of future work. In summary, this thesis has characterised a cost-effective and accessible HB-type PBR, demonstrated its application for protein expression, and explored methods to improve cell growth and protein production based on design alternations, feeding strategies and genetic engineering.

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