Publicação
Novel oscillatory flow reactors for biotechnological applications
| Resumo: | This thesis explores the biotechnological applications of two novel scale-down oscillatory flow reactors (OFRs). A micro-bioreactor (working mostly in batch) and a continuous meso-reactor systems were developed based on a 4.4 mm internal diameter tube with smooth periodic constrictions (SPC), both operating under oscillatory flow mixing (OFM). The first part is dedicated to the flow characterisation in the novel SPC geometry. Flow patterns within SPC geometry were experimentally studied using Particle Image Velocimetry (PIV) technique at different combinations of fluid oscillation frequency (x0) and amplitude centre-to-peak (x0), and afterwards used for validation of numerical simulations via Computational Fluid Dynamics (CFDs). CFD simulations were run with 2-D axisymmetric and 3-D laminar models as wells as using a turbulent Large Eddy Simulation (LES) model using Fluent (New York, USA) software. Mixing times of the micro-bioreactor were determined for batch operation at f and x0 of 0 to 20 Hz and 0-3 mm, respectively, and correlated using a newly defined mixing coefficient (km). The control of fluid dispersion in the novel SPC geometry was studied for continuous operation of both the micro-bioreactor and the meso-reactor at different combinations of f, x0 and fluid net flow rates (v). Macroscopic flow patterns were studied through the residence time distribution (RTD) and the non-ideal tracer response was modelled by four single-phase flow models, allowing the prediction of conversion ( X ) in the novel SPC tube geometry. Further RTD experiments were performed in the presence of a steady, continuous flow rate (at various values of v) and their results were compared with those obtained from CFDs simulations. Flow patterns within this novel SPC geometry were found to be very dependent of both x0 and f. In particular, km, RTD and X have demonstrated to be manipulated by the OFM conditions, as a result of a controlled fluid convection and dispersion within the SPC tube through vortex rings detachment. It is possible to drive the macroscopic flow patterns within both the micro-bioreactor and the meso-reactor towards the ideal flow cases of plug flow reactor (PFR) or completely back-mixed reactor (or a continuous stirred tank reactor, CSTR), being the convection maximized in relation to fluid dispersion mainly at smooth OFM conditions (i.e. x0 ≤ 1 mm and f ≤ 10 Hz). A 2-D axisymmetric laminar model was found to match the flow patterns at small values of f and x0 (where flow has demonstrated to match the PFR) while a 3-D laminar model was required to simulate non-axisymmetric flow patterns (as those found in a CSTR). The 3-D laminar model was highly grid-dependent, but numerical simulations with LES were found to overcome such grid dependency. Amongst the four single-phase models used in the modelling of macroscopic flow patterns by means of the analysis of RTD results, the tanks-in-series model with backflow is highly recommended due to the physical analogy with the SPC geometry (several interconnected stages – the cavities) and for considering the existence of a backflow rate, G, between the cavities. The second part of this work is focused on exploring both the micro-bioreactor and the meso-reactor in three main biotechnological applications: i) aerobic and anaerobic growth of Saccharomyces cerevisiae in the micro-bioreactor; ii) biotechnological production/screening of γ-decalactone in the micro-bioreactor; iii) dilution refolding of lysozyme for batch (micro-bioreactor) and continuous (meso-reactor) operation. Beforehand, mass transfer within the micro-bioreactor was studied by assessing the oxygen mass transfer rates in a gas-liquid system. The effect of f and x0 on the oxygen mass transfer coefficient (kLa) and on the gas hold-up (ε) were studied at a fixed gas flow rate vgas of 0.28 mL/min. An empirical correlation was developed for kLa and related with the flow patterns observed by PIV and numerically simulated with CFDs. Gas-liquid mass transfer in the micro-bioreactor was shown to be enhanced in relation to other scale-down systems, as values of kLa up to 0.05 s-1 were obtained through OFM (f = 0 - 20 Hz and x0 = 0 - 3 mm) at a small value of vgas = 0.28 mL/min. Such improved oxygen mass transfer was suggested to be responsible for an 83 % improvement of yield of biomass growth on glucose (YX/S), obtained in the aerobic growth of S. cerivisiae in comparison with the value of YX/S obtained for a stirred tank reactor (STR). Also the 50 % reduction of the time needed for maximum γ-decalactone production with the strictly aerobic yeast Yarrowia lipolytica suggested improved mass transfer rates in the four-phase system as result of an improved contact between the different phases. It has been shown that the reciprocating nature of OFM (backflow) enhances the interaction between fluid elements. This lead to the conclusion that both the microbioreactor and the meso-reactor present design limitations for lysozyme dilution refolding, mainly when applied to continuous refolding (with the meso-reactor). In fact, an intensive protein aggregation was observed, leading to the suggestion that the meso-reactor could be used as a scale-down system for production of bio-aggregates and nano-particles. In summary, the two novel scale-down platforms are ready to contribute to accelerate the bioprocess design, by allowing the running of highthroughput screening experiments at reproduced and well- ontrolled conditions. |
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| Autores principais: | Reis, N. |
| Ano: | 2006 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | This thesis explores the biotechnological applications of two novel scale-down oscillatory flow reactors (OFRs). A micro-bioreactor (working mostly in batch) and a continuous meso-reactor systems were developed based on a 4.4 mm internal diameter tube with smooth periodic constrictions (SPC), both operating under oscillatory flow mixing (OFM). The first part is dedicated to the flow characterisation in the novel SPC geometry. Flow patterns within SPC geometry were experimentally studied using Particle Image Velocimetry (PIV) technique at different combinations of fluid oscillation frequency (x0) and amplitude centre-to-peak (x0), and afterwards used for validation of numerical simulations via Computational Fluid Dynamics (CFDs). CFD simulations were run with 2-D axisymmetric and 3-D laminar models as wells as using a turbulent Large Eddy Simulation (LES) model using Fluent (New York, USA) software. Mixing times of the micro-bioreactor were determined for batch operation at f and x0 of 0 to 20 Hz and 0-3 mm, respectively, and correlated using a newly defined mixing coefficient (km). The control of fluid dispersion in the novel SPC geometry was studied for continuous operation of both the micro-bioreactor and the meso-reactor at different combinations of f, x0 and fluid net flow rates (v). Macroscopic flow patterns were studied through the residence time distribution (RTD) and the non-ideal tracer response was modelled by four single-phase flow models, allowing the prediction of conversion ( X ) in the novel SPC tube geometry. Further RTD experiments were performed in the presence of a steady, continuous flow rate (at various values of v) and their results were compared with those obtained from CFDs simulations. Flow patterns within this novel SPC geometry were found to be very dependent of both x0 and f. In particular, km, RTD and X have demonstrated to be manipulated by the OFM conditions, as a result of a controlled fluid convection and dispersion within the SPC tube through vortex rings detachment. It is possible to drive the macroscopic flow patterns within both the micro-bioreactor and the meso-reactor towards the ideal flow cases of plug flow reactor (PFR) or completely back-mixed reactor (or a continuous stirred tank reactor, CSTR), being the convection maximized in relation to fluid dispersion mainly at smooth OFM conditions (i.e. x0 ≤ 1 mm and f ≤ 10 Hz). A 2-D axisymmetric laminar model was found to match the flow patterns at small values of f and x0 (where flow has demonstrated to match the PFR) while a 3-D laminar model was required to simulate non-axisymmetric flow patterns (as those found in a CSTR). The 3-D laminar model was highly grid-dependent, but numerical simulations with LES were found to overcome such grid dependency. Amongst the four single-phase models used in the modelling of macroscopic flow patterns by means of the analysis of RTD results, the tanks-in-series model with backflow is highly recommended due to the physical analogy with the SPC geometry (several interconnected stages – the cavities) and for considering the existence of a backflow rate, G, between the cavities. The second part of this work is focused on exploring both the micro-bioreactor and the meso-reactor in three main biotechnological applications: i) aerobic and anaerobic growth of Saccharomyces cerevisiae in the micro-bioreactor; ii) biotechnological production/screening of γ-decalactone in the micro-bioreactor; iii) dilution refolding of lysozyme for batch (micro-bioreactor) and continuous (meso-reactor) operation. Beforehand, mass transfer within the micro-bioreactor was studied by assessing the oxygen mass transfer rates in a gas-liquid system. The effect of f and x0 on the oxygen mass transfer coefficient (kLa) and on the gas hold-up (ε) were studied at a fixed gas flow rate vgas of 0.28 mL/min. An empirical correlation was developed for kLa and related with the flow patterns observed by PIV and numerically simulated with CFDs. Gas-liquid mass transfer in the micro-bioreactor was shown to be enhanced in relation to other scale-down systems, as values of kLa up to 0.05 s-1 were obtained through OFM (f = 0 - 20 Hz and x0 = 0 - 3 mm) at a small value of vgas = 0.28 mL/min. Such improved oxygen mass transfer was suggested to be responsible for an 83 % improvement of yield of biomass growth on glucose (YX/S), obtained in the aerobic growth of S. cerivisiae in comparison with the value of YX/S obtained for a stirred tank reactor (STR). Also the 50 % reduction of the time needed for maximum γ-decalactone production with the strictly aerobic yeast Yarrowia lipolytica suggested improved mass transfer rates in the four-phase system as result of an improved contact between the different phases. It has been shown that the reciprocating nature of OFM (backflow) enhances the interaction between fluid elements. This lead to the conclusion that both the microbioreactor and the meso-reactor present design limitations for lysozyme dilution refolding, mainly when applied to continuous refolding (with the meso-reactor). In fact, an intensive protein aggregation was observed, leading to the suggestion that the meso-reactor could be used as a scale-down system for production of bio-aggregates and nano-particles. In summary, the two novel scale-down platforms are ready to contribute to accelerate the bioprocess design, by allowing the running of highthroughput screening experiments at reproduced and well- ontrolled conditions. |
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