Rutley, James David; (2019) Evaluation of Single-Use Bioreactors for Rapid Development of Industrial Fermentation Processes. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Microbial fermentation and whole cell biocatalysis have long been used in an industrial context for the generation of commercially valuable biomolecules. There is significant further potential if they can be made as economically attractive as competing chemical process routes. Improvements in genetic engineering techniques such as those pioneered in the area of synthetic biology, have provided access to novel products including therapeutic proteins and enzymes. In order to capitalise on these advances, there remains a requirement for rapid bioprocess development and scale up. Here, single use bioreactors are of interest due to the advantages over traditional stainless steel technologies. These include reduced need for validation and turnaround times, a reduction in capital expenditure and increased facility flexibility. To date, however, they have not been thoroughly investigated for use with microbial expression systems. The aim of this thesis is to; (i) evaluate the oxygen transfer capabilities of different single-use bioreactors with a view to determining suitability for microbial fermentation, and (ii) to define appropriate scale-up bases from high throughput microwell to laboratory and pilot scale bioreactors. Initial work focused on the characterisation, optimisation and scale-up of a whole cell P450 monooxygenase bioconversion in Escherichia coli. Three rounds of optimisation using a Design of Experiments (DoE) methodology resulted in a 3.3 fold increase in the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Results from the 96 deep square well (DSW) plates were then scaled-up to a traditional, pilot scale stirred tank bioreactor, increasing titres 25 times over a 3000-fold scale increase. Peak oxygen demands of 25.8 mmolL-1min-1 were shown, with clear differences in oxygen consumption as a result of feeding and bioconversion during fermentation. The inability of microwell systems to support high biomass concentrations and oxidative bioconversion was also demonstrated. In addition, these studies helped provide fundamental insights into the mechanism for oxygen utilisation during microbial whole cell bioconversions. Prioritisation of oxygen utilisation for biomass accumulation over supplementary cellular activities such as bioconversion was seen in all cases. In some cases, oxygen demand as a result of growth was approximately 4 times greater than other contributions. This had previously been hypothesised in literature but not demonstrated. In order to better characterise oxygen mass transfer in microwell plate geometries, an improved method for quantification of the volumetric oxygen mass transfer coefficient (kLa) and oxygen uptake rate (OUR), based on the dynamic gassing out method, was subsequently developed. This method determines oxygen mass transfer parameters (kLa and OUR) during a fermentation to provide more representative values for OUR. Models for OUR and kLa were built in 24 DSW plates with maximum values of over 600 mgO2L-1h-1 and 103.5 h-1 respectively. The established models enable equivalent operating conditions for the different plate geometries to be determined. The applicability of two commercially available single use bioreactors (the Ambr®250 and the XDR-10) for microbial fermentation was evaluated, using a traditional pilot scale STR for comparison. This included building models which consider a number of factors likely to influence oxygen mass transfer simultaneously, as opposed to the empirical correlations which have been developed traditionally. The Ambr®250 was demonstrated as having similar oxygen mass transfer capability to the STR across the majority of the experimental ranges, reaching maximum kLa values of > 600 h-1. Analysis of a large number of industrial microbial fermentations (approximately 300) demonstrated that the Ambr®250 is capable of supporting microbial fermentation and bioconversion (or recombinant protein expression) in each, where the XDR-10 would not be suitable. After demonstrating the applicability of the Ambr®250 system for industrial microbial fermentation, a modelling tool was developed in the Python programming language capable of evaluating the cost and resource requirements of an Ambr®250 bioprocess development run. Preliminary sensitivity analysis highlights labour as the main influence on cost, followed by the replacement of single use bioreactors, each responsible for more than one third of the total run cost. Overall this work has established original, quantitative insights into oxygen utilisation in microbial expression systems and established engineering criteria for the selection and use of single-use bioreactor technologies for microbial cultivation. The methodologies developed here are considered generic and applicable to other expression systems with high specific oxygen demands such as yeast and heterotrophically cultured microalgae.

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