Binti Suhaili, Nurashikin; (2017) Characterisation of biocatalyst production within an integrated biorefinery context. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

With the emerging interest in integrated biorefinery concepts, there is a need to identify and develop profitable product streams and ensure the utilisation of as many waste streams as possible. Early stage bioprocess development for these processes can be facilitated by the use of high throughput bioreactor platforms that enables rapid, quantitative and scalable data acquisition. This thesis aims to establish high throughput methodologies for the production and characterisation of industrial biocatalysts within an integrated biorefinery context. Specifically, the work focuses on the production of the CV2025 ω-Transaminase (CV2025 ω-TAm) in Escherichia coli BL21 (DE3) using sugar beet vinasse, a bioethanol waste stream, as a fermentation feedstock. The high throughput platform to be explored is a 24-well, controlled microbioreactor (MBR) that provides individual monitoring and control of process parameters at the well level. Initially, batch E. coli BL21 (DE3) fermentations expressing CV2025 ω-TAm were established in the controlled MBR using a synthetic medium to provide benchmark data on cell growth and enzyme expression. These cultures indicated a good degree of monitoring and control with respect to process parameters as well as culture reproducibility across the wells. Significant enhancements in relation to maximum biomass concentration (Xmax), yield of biomass on substrate (YX/S) and CV2025 ω-TAm specific activity of 3.7, 1.9 and 2.2-fold, respectively, were shown in the MBR compared to conventional shake flask system, also representing a 31-fold volumetric reduction. Optimisation of CV2025 ω-TAm production in the MBR showed that the best cell growth and enzyme titre was achieved with an early induction (6 h), 0.1 mM IPTG and 0.024 mmol IPTG gdcw-1, yielding enhancements in Xmax, YX/S and CV2025 ω-TAm specific activity of 1.04, 1.2 and 1.4-fold, respectively over the non-optimised cultures. Control of dissolved oxygen (DO) levels between 30 - 50% oxygen saturation had no significant impact on cell growth and CV2025 ω-TAm titre. Evaluation of vinasse as a fermentation feedstock for CV2025 ω-TAm production has led to several novel findings. Characterisation of vinasse showed that the feedstock comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 (DE3) cell growth and CV2025 ω-TAm production were feasible in cultures using 17 to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The D-galactose in vinasse was shown to facilitate auto-induction of the pQR801 plasmid leading to comparable CV2025 ω-TAm expression as obtained in IPTG-induced cultures. Assessment of different vinasse pre-processing options confirmed the relevance of the dilution step in reducing polyphenol concentrations to below inhibitory levels. Moreover, the use of pasteurised vinasse was found to be promising for large scale applications. Further medium optimisation studies in the MBR showed the benefit of supplementing vinasse with specific media components. Supplementation of diluted vinasse medium with 10 g L-1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, Xmax, CV2025 ω-TAm volumetric and specific activity, respectively, over those achieved in non-supplemented cultures. Additionally, the CV2025 ω-TAm titre attained here represented 81% of that obtained using an optimised synthetic medium. Investigation into the metabolic preferences of E. coli BL21 (DE3) when grown on a complex carbon source like vinasse showed the sequential metabolism of D-mannitol before glycerol utilisation, which was followed by the simultaneous metabolism of glycerol, D-xylitol, D-dulcitol and acetate thereafter. Finally, scale-up of the optimal conditions for CV2025 ω-TAm production using both synthetic and vinasse-based media, from the controlled MBR to a 7.5 L stirred tank reactor (STR) was shown based on matched kLa values and specific aeration rates. Results showed a good reproducibility with respect to cell growth, substrate consumption and CV2025 ω-TAm production between the scales, representing a 769-fold volumetric scale translation. The feasibility of further intensification of CV2025 ω-TAm production in STR at higher kLa values using both synthetic and vinasse-based media was also demonstrated leading to enhancements of 1.4 and 1.9-fold in enzyme titre, respectively. Overall, this work has established high throughput methodologies for the characterisation, optimisation and scale-up of industrial biocatalyst production. The approach was demonstrated within the context of an integrated sugar beet biorefinery. However, the utility of the high throughput approach is considered generally applicable across the industrial biotechnology sector.

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