Academic literature on the topic 'Stirred tank bioreactor'

Gas–liquid mixing equipment is applied widely in chemical industries. Many research available in literature have been carried out on this topic, but the majority of the studies deal with fluids of water-like viscosity. However, in practical industrial process, such as in pharmaceutical engineering, the process fluid exhibits non-Newtonian characteristics. The main objective of this study is to investigate the rheological properties influence of a non-Newtonian shear thinning fluid as representative of a fermentation broth. The study will involve two experimental setup, i.e. stirred tank bioreactor (STB) and orbitally shaken bioreactor (OSB), for comparison purposes. The hydrodynamic behavior of the non-newtonian fluid will be investigated in both bioreactors, with the aim to understand the main differences between such fluid and water. Furthermore, both systems will be analyzed under the process aspect, with particular attention to gas-to-liquid mass transfer. The goal of the project is to compare the widely-employed STB with the new OSB, considered more suitable for low shear stresses applications.

Resistance to antibiotics by microbial pathogens continues to be a major global health problem. Treatment of bacterial infections is becoming increasingly complex and expensive. Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is affected by antibiotic resistance. In South Africa, the Western Province is the worst affected, with an increasing incidence of both multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis. Both resistant forms of TB increase the length of treatment to almost 24 months and cost by as much as 1400 times that of regular anti-tubercular chemotherapy. A potential solution to this problem is the discovery of new drugs, which can be obtained from natural sources. Actinomycetes are good sources of these drugs, with over 45% of current medicines derived from these bacteria. The actinobacterium Streptomyces polyantibioticus SPRT (SPRT) was locally isolated and first described by Le Roes (2006). It has been shown to produce bioactive molecules active against a range of bacteria, including compounds (drugs) that have anti-tubercular properties. One of the anti-tubercular molecules was identified as 2,5-diphenyloxazole (DPO). DPO is currently used as a component of scintillation fluid for its luminescent properties and is synthesised chemically in industry. SPRT is the only reported biological source of DPO, it is however not yet produced commercially via a biological route. The present study was performed to inform future process development of DPO production from SPRT. An investigation into the growth and production of antimicrobial compounds from submerged cultures of SPRT in shake flasks, and scale-up of the process into a laboratory stirred tank bioreactor (STR) was done in the present study. The work focused on obtaining growth kinetics and suitable operating conditions for cultivation. Characterisation of the growth profile of SPRT and determination of the kinetic growth parameters was carried out. Additionally, the antimicrobial production phases, and factors influencing their production was investigated. It was determined that the most reliable method of measuring biomass concentration was by dry cell gravimetric measurement of whole shake flasks following vacuum filtration, as it best suited the non-homogenous filamentous nature of SPRT.

A recombinat Chinese Hamster Ovary (rCHO) cell line designated as CHO SEAP was utilized in this investigation to optimize protein production. Two bench top stirred-tank bioreactors, namely a pitched-blade and a packed-bed basket bioreactor, were utilized for a comparative study to determine which bioreactor would produce the best results in terms of protein production. The objective of this research project was to provide basic data that shows cells cultured in a packed-bed basket bioreactor in perfusion mode will generate more protein product than cells in batch mode suspension culture with a pitched-blade bioreactor. The packed-bed bioreactor creates a homeostatic environment similar to the environment found in vivo, where waste products are constantly removed and fresh nutrients are replenished. Closed batch cultures do not provide a homeostatic environment. In batch culture systems, nutrients are depleted and waste products accumulate. The results from this experiment could help investigators involved in protein and/or vaccine production facilities select the appropriate bioreactor and mode of operation to optimize cell productivity for generation of a specific protein product. CHO cells have been used for the production of vaccines, recombinant therapeutic proteins, and monoclonal antibodies, and these cells are now the cell line of choice in the biopharmaceutical industry. Traditional vaccine production methods in egg embryos are slow and outdated, whereas roller bottle-based cell culture techniques are time consuming and have limited scalability. These limitations justify the need for development of stirred tank bioreactors. Cells cultured in a packed-bed bioreactor are not exposed to hydrodynamic forces, as is the case with pitched-blade bioreactors, allowing for maximum growth and protein expression. This mode of operation involves the constant removal of media depleted of nutrients and the addition of fresh media with more nutrients to keep the cells growing. Long run times decrease the constant need for re-seeding cells and re-establishing seed cultures, thus, reducing setup time and labor dramatically. Secreted products are automatically separated from cells in perfusion, eliminating filtration and membrane fouling. A detailed description of both modes of operation are discussed in this thesis.

Les cellules souches mésenchymateuses (CSM) interviennent de plus en plus dans le domaine de la médecine régénérative, notamment pour traiter des maladies aujourd’hui difficilement curables avec les moyens actuels. Deux verrous scientifiques limitent pourtant leur utilisation et leur commercialisation. D’une part, de grandes quantités de cellules sont nécessaires pour répondre à la forte demande médicale. D’autre part, les cellules étant elles-mêmes le médicament final, délivré chez le patient, leur qualité doit être préservée (phénotype souche, capacité de différenciation). La mise en culture de ces cellules, sur des microporteurs, en bioréacteur agité, semble répondre à ces enjeux. Cependant, une connaissance plus précise de l’impact, sur la réponse physiologique des cellules, des technologies utilisées et de l’hydrodynamique générée est nécessaire pour améliorer les lois d’extrapolation des bioréacteurs de culture de CSM. Dans ce contexte, des travaux ont été mis en œuvre pour étudier l’influence du mode d’agitation (orbital ou mécanique) sur l’attachement, l’expansion et le détachement de CSM issues de la gelée de Wharton (GW-CSM) de cordons ombilicaux, sur des microporteurs de différentes compositions. Pour contribuer à la quantification de l’expansion cellulaire, une méthode de comptage automatique in situ a été développée pour estimer le nombre de cellules par microporteur, ainsi que leur répartition, sans avoir à procéder à leur détachement. Des microporteurs commerciaux ont ensuite pu être comparés à des microporteurs synthétisés dans un laboratoire partenaire, en termes d’attachement et expansion cellulaire, ainsi que de facilité de détachement. En parallèle de ces travaux, l’impact de la conception du mobile d’agitation, en bioréacteur mécaniquement agité, sur la mise en suspension de microporteurs a été analysé. A l’issue de cette étude, une analyse dimensionnelle et des simulations CFD ont été mises en place et deux modèles reliant la fréquence minimale de juste mise en suspension (Njs) avec la géométrie du mobile d’agitation (forme, taille, position dans la cuve) et les propriétés matérielles des particules et de la phase liquide ont été proposés. Une stratégie d’optimisation des paramètres géométriques d’un mobile en minibioréacteur, dédié à la culture de CSM sur microporteurs, a été mise en place, à partir de paramètres caractérisant les contraintes hydromécaniques perçues par la phase solide, judicieusement choisis et intégrés lors des simulations CFD. Selon un plan d’expérience, et les résultats extraits des simulations, des surfaces de réponse ont été construites et une optimisation multi-objective a été réalisée afin de déterminer la géométrie minimisant les contraintes perçues par les particules, et donc par les cellules adhérées. Des cultures de GW-CSM en minibioréacteurs équipés de différents mobiles ont finalement été validées, avec une comparaison préliminaire de l’impact de ces géométries sur l’expansion cellulaire<br>Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion

Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.

Xanthan is a well-known extracellular polysaccharide, produced by a Gram negative bacterium Xanthomonas campestris (X. campestris) under aerobic conditions. Solutions of xanthan exhibit high viscosities and non-Newtonian behaviour even at low concentrations. This biopolymer has a wide range of valuable commercial and industrial applications, for example; it can be used as a food thickening agent and a stabilizer in some other industries. Traditionally the production of xanthan has predominantly been performed in stirred tank fermenter (STR). This study sought to compare the cultivation of the bacterium, X. campestris for the production of the viscous biopolymer xanthan gum in two different reactor systems, a novel oscillatory baffled reactor (OBR) and the conventional industry workhorse, the stirred tank reactor (STR). Overall biopolymer production occurred at similar rates in the well stirred and aerated STRs, albeit at the cost of higher energy inputs for mixing and aeration. Despite much previous literature promoting the use of the OBR for transporting and reacting very viscous systems, this was the first actual study attempting to investigate the use of the OBR for a highly viscous non-Newtonian fermentation process. The experimental results show that xanthan production was similar in the OBR than in the STR, the OBR is however readily suitable for the cultivation of xanthan. The probable reasons for the inability of the OBR to match the production rates of the STR may well lie in the complex nature of this fermentation process. Unlike a previous study on pullulan production (Gaidhani 2004) where the OBR outperformed the STR, X. campestris initially needs high oxygen transfer rates and the OBR, although it provides good bulk mixing and low energy consumption, seemed unable to equal the STR in this respect, especially in a very viscous system. The result shows that xanthan production in the OBR was similar to the equivalent process in the STR. In order to attempt to improve the OBR a number of technical modifications were made including a novel sparger design to improve gas dispersal. These were not successful in improving xanthan production. Similarly, attempts to achieve improvements via wider amplitude ranges led to damage to the equipment. The conclusion was that significant improvements to the physical robustness of the OBR were necessary before it could be successfully used to process highly viscous bio-fluids.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.

Polycyclic aromatic hydrocarbons (PAHs) do not dissolve easily in water, due to their hydrophobic properties. PAHs are unavailable to most aromatic compound degrading organisms, due to these properties. In this study, a biosurfactant producing culture enhancing dissolution of PAHs was isolated, to make them bioavailable. The culture was introduced to the system to improve the dissolution of PAHs and degrade the PAHs thereafter. The aim of the study was to use a strategy with a biofilm process, subsequent to a continuous stirred tank bio-reactors (CSTRs) to successfully remove PAHs from water, with microorganisms that can degrade these pollutants. The open system could easily be controlled and set to optimum conditions, stimulating the growth of PAH degraders. The feed rate and influent concentration can be controlled and the system can easily be cleaned. Biodegradation was achieved, using optimum conditions obtained from the conducted batch studies in a CSTR process ensuring a feasible biodegradation process. Two cultures, Pseudomonas aeruginosa and microbial consortia, were used during the biosurfactant production and PAHs degradation preliminary batch studies. The biosurfactants produced, were identified as Lipopeptides and degradation results indicated great degradation of fluoranthene and triphenylene with a mixed culture consortium present in the system. 90.1% of fluoranthene and 79.6% of triphenylene was degraded after 22 d of incubation in the batch system. Degradation of fluoranthene was studied using biosurfactants and microbial consortium in a three-stage continuous flow system. Reactor 2A fluoranthene influent (60.89%) was degraded, 70.02% of Reactor 2B fluoranthene influent was degraded and 77.17% of biofilm tank fluoranthene influent was degraded. Kinetic studies were conducted, using a Monod model to describe the substrates degradation for batch systems. The highest degradation rate for fluoranthene was determined to be 0.29 h-1 and for triphenylene was 0.13 h-1 with half saturation values of 991.84 mg/L and 451 mg/L respectively, indicating that fluoranthene was degraded faster than triphenylene, when incubated for 22 d. The study demonstrated that biosurfactant production and biodegradation of fluoranthene can be achieved in an open CSTR system, as much as it can be done in a batch system. The biological remediation of PAHs in wastewater plants can be introduced and applied for wastewaters rich, with PAHs.<br>Dissertation (MSc)--University of Pretoria, 2017.<br>Chemical Engineering<br>MSc<br>Unrestricted