Search

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cells as precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity.

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (NJS) and associated specific power input (P/V)JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

BACKGROUND Traditional large‐scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface‐to‐volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two‐phase system for expansion and non‐enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein‐rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non‐enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION The prospect of a temporary microcarrier that can be used to expand cells and then ‘disappear’ for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality.

BACKGROUND 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) that occur at the larger scale in a bench‐scale two‐compartment reactor [plug flow reactor (PFR) + stirred tank reactor (STR)] using the cadaverine‐producing recombinant Corynebacterium glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT. The new scale‐down strategy developed here studied the effect of increasing the magnitude of fermentation gradients by considering not only the average cell residence time in the PFR (τPFR), but also the mean frequency at which the bacterial cells entered the PFR (fm) section of the two‐compartment reactor. RESULTS On implementing this strategy the cadaverine production decreased on average by 26%, 49% and 59% when the τPFR was increased from 1 to 2 min and then 5 min respectively compared to the control fermentation. The carbon dioxide productivity was highest (3.1‐fold that of the control) at a τPFR of 5 min, but no losses were observed in biomass production. However, the population of viable but non‐culturable cells increased as the magnitude of fermentation gradients was increased. The new scale‐down approach was also shown to have a bigger impact on fermentation performance than the traditional one. CONCLUSION This study demonstrated that C. glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT physiological response was a function of the magnitude of fermentation gradients simulated. The adaptations of a bacterial cell within a heterogeneous environment ultimately result in losses in fermentation productivity as observed here. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukaemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells which remains a major translational and commercial bottleneck due to the manual, small-scale and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared to T-flasks and culture bags. In this study, we demonstrate, that not only can T-cells be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby higher specific power inputs (P/M W kg-1 ), the higher the final viable T-cell density; i. e., a cell density of 4.65 ± 0.24 x 106 viable cells ml-1 obtained at the highest P/M of 74 x 10-4 W kg-1 in comparison to 0.91 ± 0.07 x 106 viable cells ml-1 at the lowest P/M of 3.1 x 10-4 W kg-1 . We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby the cells grow better under higher agitation speeds whilst maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications. This article is protected by copyright. All rights reserved.

Human Mesenchymal Stem Cells (hMSCs) are advancing through clinical development with the first allogeneic adult hMSC therapy receiving approval in Europe. To enable successful large-scale manufacture of hMSC therapies, increased product consistency and yield, and a reduced batch-to-batch variation must be achieved. This paper addresses ways to reduce variation by controlling the processing conditions, in particular the dissolved oxygen concentration (dO2), and the culture medium. Bone marrow derived hMSCs were cultured in DASGIP DASbox bioreactors on Plastic P-102 L microcarriers in FBS-containing and serum free (SFM) media at various dO2 values from 100% to 10%, experiencing the same dO2 value throughout the culture process. The superior control of pH and dO2 in the bioreactor led to improved performances compared to poorly controlled spinner flasks, particularly at reduced dO2 concentrations. At 25% dO2, there was a 300 % increase in the BM-hMSC yield in the bioreactor across the two donor BM-hMSCs in SFM compared to FBS-containing medium. Overall, the process yield increased by an average of around 500% for both donors under controlled conditions in SFM at 25% dO2 in the bioreactor compared to the poorly controlled expansion at atmospheric conditions in FBS-containing medium in spinner flasks. Process control significantly reduced the BM-hMSC variation in yield from 79.1% in FBS-containing medium in spinner flasks to < 15% in controlled SFM bioreactor culture.

International audience; Cellulases for bioethanol production are mainly made by fed-batch fermentation using a filamentous fungus, Trichoderma reesei. Agitation at different scales impacts on morphology, rheology and growth rate and can be correlated by EDCFεmax. Typically, EDCFεmax is much smaller at commercial scale and fungal size, viscosity and growth rate are greater. Here, to increase understanding, continuous culture in 3 L bioreactors using two EDCFεmax values were undertaken. The higher EDCFεmax decreased the cellulase production (concentration, 21%; specific production rate, 24%; protein yield, 20%) whilst proteomic analysis showed, at an intracellular level, a decrease of cellulase and hemicellulase synthesis. An increase of stress proteins also occurred, which may help cells to limit the impact of fluid dynamic stress. Also, cellulase production during continuous culture at the bench varied with EDCFεmax similarly to that between bench and commercial scale during fed-batch culture.

Human mesenchymal stem/stromal cells (hMSCs) present a key therapeutic cellular intervention for use in cell and gene therapy (CGT) applications due to their immunomodulatory properties and multi-differentiation capability. Some of the indications where hMSCs have demonstrated pre-clinical or clinical efficacy to improve outcomes are cartilage repair, acute myocardial infarction, graft versus host disease, Crohn's disease and arthritis. The current engineering challenge is to produce hMSCs at an affordable price and at a commercially-relevant scale whilst minimising process variability and manual, human operations. By employing bioreactors and microcarriers (due to the adherent nature of hMSCs), it is expected that production costs would decrease due to improved process monitoring and control leading to better consistency and process efficiency, and enabling economies of scale. This approach will result in off the shelf (allogeneic) hMSC-based products becoming more accessible and affordable. Importantly, cell quality, including potency, must be maintained during the bioreactor manufacturing process. This review aims to examine the various factors to be considered when developing a hMSC manufacturing process using microcarriers and bioreactors and their potential impact on the final product. As concluding remarks, gaps in the current literature and potential future areas of research are also discussed.

The impact of agitation rate and sparged aeration on BM-hMSC expansion in conventional stirred tank bioreactors was assessed. It was found that a decrease in impeller speed to below NJS caused sampling difficulties, clumping and an increase to ~2 NJS decreased the growth rate though an intermediate value of ~1.3 NJS did not. Additionally, over this range of agitation intensities, cell quality remained unchanged post-harvest suggesting that poor growth performance at the highest speed was due to a failure of the cells to attach efficiently to microcarriers rather than damage to the cells due to fluid dynamic stress. Further it was shown that direct aeration of the culture medium both with and without Pluronic F68 via a sparger at NJS was detrimental to BM-hMSC growth. Again, this reduction in growth seems to be associated with poor attachment rather than cell damage, which due to the mechanism of PluronicTM F68 reducing the cell hydrophobicity and thus the affinity of the BM-hMSCs to attach to the microcarrier, leads to a poorer performance in the presence of the surfactant. Certain post-harvest quality characteristics are also detrimentally impacted compared to headspace aeration. This problem is discussed in terms of the need to facilitate future large-scale process development where headspace aeration at NJS may not be sufficient to meet culture needs at higher cell densities.

Human mesenchymal stem cells (hMSCs) are a key candidate for advanced cell therapies with numerous clinical trials investigating their potential to treat acute and chronic indications. However, important translational and manufacturing challenges need to be addressed to improve our capability for scalable production of fully functional cells. In this study, we have demonstrated, both qualitatively and quantitatively, the ability of bone marrow-derived hMSCs to migrate from one microcarrier to another, and, to populate fresh microcarriers when added into suspension culture. Additionally, we have shown that compared to inoculating a culture with cells in free suspension, inoculating 10% of near-confluent microcarriers from an initial seed microcarrier culture resulted in an increase in the cell growth rate and overall cell yield and a significant reduction in the lag phase. These findings were consistent across cells from three different BM-hMSC donors and across different culture medium conditions, foetal bovine serum-supplemented medium, human platelet lysate-supplemented medium and serum-free medium. This new cells-on-beads inoculation method is an effective means of process intensification with the potential to decrease manufacturing times and potentially costs of hMSC-based therapies.

Cell and gene-based therapies represent a novel therapeutic modality that has the potential to provide a treatment option for a range of medical conditions. There are, however, numerous processing and manufacturing challenges that must be addressed before such therapies are considered commercially and clinically viable. A significant challenge associated with the manufacture of such therapies is ensuring cell quality and the product’s critical quality attributes are retained throughout the entire bioprocess. Biopreservation is an important aspect of cell and gene-based therapy bioprocessing, which enables the development of cell banks. It increases process flexibility by providing a shelf-life to the product, enables the storage of intermediates and provides breakpoints within the process. In this article, we summarize the advances and challenges associated with biopreservation of cell and gene therapies.

BACKGROUND Human mesenchymal stem/stromal cells (hMSCs) are at the forefront of regenerative medicine applications due to their relatively easy isolation and availability in adults, potential to differentiate and to secrete a range of trophic factors that could determine specialised tissue regeneration. To date, hMSCs have been successfully cultured in vitro on substrates such as polystyrene dishes (TCPS) or microcarriers. However, hMSC sub-cultivation and harvest typically employs proteolytic enzymes that act by cleaving important cell membrane proteins resulting in long-term cell damage. In a process where the cells themselves are the product, a non-enzymatic and non-damaging harvesting approach is desirable. RESULTS An alternative system for hMSC expansion and subsequent non-enzymatic harvest was investigated here. A liquid/liquid two-phase system was proposed, comprising a selected perfluorocarbon (FC40) and growth medium (DMEM). The cells exhibited similar cell morphologies compared with TCPS. Moreover, they retained their identity and differentiation potential post-expansion and post-harvest. Further, no significant difference was found when culturing hMSCs in the culture systems prepared with either fresh or recycled FC40 perfluorocarbon. CONCLUSIONS These findings make the FC40/DMEM system an attractive alternative for traditional cell culture substrates due to their ease of cell recovery and recyclability, the latter impacting on overall process costs. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Filamentous fungi have a complex morphology that induces fermentation process development issues, as a consequence of viscosity increase and diffusion limitations. In order to better understand the relationship between viscosity changes and fungus morphology during fermentations of Trichoderma reesei, an accurate image analysis method has been developed to provide quantitative and representative data for morphological analysis. This method consisted of a new algorithm called FACE that allowed sharp images to be created at all positions, segmentation of fungus, and morphological analysis using skeleton and topological approaches. It was applied and validated by characterizing samples of an industrial strain of Trichoderma reesei that had or had not been exposed to an extreme shear stress. This method allowed many morphological characteristics to be identified, among which nine relevant criteria were extracted, regarding the impact of shear stress on the fungus and on the viscosity of the fermentation medium.

BACKGROUND: Application of perfluorocarbon based liquid/liquid two-phase systems for cell culture expansion has been investigated at small scale for more than 30 years and it has been established that such systems are able to support the survival of a variety of cell lines. Application of drops in liquid/liquid dispersions as temporary microcarriers is an exciting prospect as it enables adherent cells to be grown in stirred bioreactors, without the need to use enzymatic dissociation methods to harvest the cells. RESULTS: Two aspects of scaling up of perfluorocarbon/cell culture medium dispersions were investigated: (i) the effect of processing conditions on drop size/interfacial area; and (ii) the kinetics of separation of a stagnant dispersion. The processing conditions to produce the stable 'liquid microcarriers' with average drop size between 150 and 220μm have been established. Separation of dispersion into two continuous systems requires complete removal of proteins from the perfluorocarbon/cell culture medium interface. CONCLUSIONS: The correlation relating average drop size to the energy input and physical properties of both phases was developed and the method of separation of stable perfluorocarbon/cell culture medium dispersion was established. As the perfluorocarbon does not deteriorate during cell expansion and subsequent separation followed by sterilization, it could be re-used, making application of such systems at large scale very attractive and economical.

A quantitative methodology for particle suspension assessment is presented. A new parameter, fmov/tot, the ratio of the mean number of moving particles to the total number of particles, is introduced to evaluate the minimum speed required to just suspend solids. This approach is tested to investigate the impact of impeller clearance on the minimum impeller speed, Njs, in a vessel when using a radial flow Rushton turbine. Flow patterns and power numbers obtained experimentally and computationally support the suspension findings. Image analysis is an appropriate method for determining Njs. Lowering the impeller clearance reduces the speed required for particle suspension with a change of flow pattern from a radial discharge with two loops to a single loop scouring the vessel base. The power number also falls markedly at the two-to-one loop transition as does the strain rate near the base.

Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Chimeric antigen receptor T-cell (CAR-T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR-T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR-T cells from multiple donors in an automated stirred-tank bioreactor. The authors successfully produced functional human CAR-T cells from multiple donors under dynamic conditions in a stirred-tank bioreactor, resulting in overall cell yields which were significantly better than in static T-flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR-T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 cells ml-1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T-flasks and gas-permeable culture bags). Importantly, engineered T-cells post-expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR-T cell manufacture in stirred-tank bioreactors and the findings present significant implications and opportunities for larger-scale allogeneic CAR-T production.

Stirred-tank reactors are used throughout the process industry. They are not only applied in the chemical industry, but also in newer industrial sectors, such as biologics, pharmaceuticals, nanotechnology, and regenerative medicine. Certain features have not changed for many years, but the understanding of the physical processes occurring within has greatly increased. Since knowledge of the interaction between the physics of mixing, single- or multiphase, and the underlying science is critical, the physical processes of mixing/stirring are considered as they developed over the last ∼75 years.

International audience; Scale-up of aerobic fermentation fungi processes still remains a challenging issue for the biotechnology industry. This difficultyarises due to the complex interactions between operating conditions (agitation, aeration, etc.), the physicochemical state of the broth (viscosity, the dissolved oxygen concentration, etc.) and the biology of fungi (growth, production, morphology, etc.). Because of their size, filamentous fungi are affected by fluid dynamic stresses but quantification of this complex parameter is a difficult task. In general, indirect criteria are used for the effect of fluid dynamic stresses on scale-up (tip speed, power draw or the energy dissipation/circulation function (EDCF)). In order to better understand the impact of such criteria on the fermentation of the fungus Trichoderma reesei, a wide range of agitation conditions has been explored. The morphology of T. reesei fungus, its specific growth rate and the rheological properties of the broth have all been measured both at bench scale (~ 2.5 L) and for the first time, at a typical commercial scale. These three aspects of the fermentation at both scales were then compared with respect to tip speed, specific power and EDCF. This work has shown that tip speed as a correlator of any of these parameters is totally ineffective whilst the EDCF is clearly the best for extrapolating laboratory data to the commercial scale

Science and medicine has revealed the potential of cell therapies; however, for this to be realised, scalable and robust manufacturing processes must be developed for the reproducible production of clinically-relevant numbers of fully functional stem cells. It is well established that for all products made either by cell culture or fermentation, the process must be defined with equally defined critical quality attributes (CQAs) that can be monitored and controlled to ensure product safety as well as efficacy. Human mesenchymal stem cells need to be attached to a surface to grow and cannot be adapted to suspension culture. For allogeneic cell therapy processes, scale-up approaches are more likely to be employed. As well as the physical challenge of removing the cells from the microcarriers, there remains a challenge in demonstrating comparability of cell CQAs with the monolayer process, which will be essential to effectively scale up the process.

Two series of reproducible fed-batch bench scale cultures have been undertaken, one series simulating the impact of spatial variations in pH and nutrients as found at commercial scale on performance, the other, the impact of fluid dynamic stresses associated with agitation. The first was unsuccessful because, somewhat surprisingly, the use of a peristaltic pump to circulate cells and medium through different spatial environments always led to a similar reduction in culture time and resulting product titre compared to uncirculated controls. This fall was sufficient to essentially mask other effects. In the second, even at maximum specific energy dissipation rates up to ∼160 times > with laminar extensional flow and ∼25 times > with turbulent flow compared to typical commercial conditions, no significant effects were observed on cell growth and viability. Most importantly, in all of the cases studied, product quality was unaffected compared to controls. In addition, it is suggested that because of the possibility of cell line specific behaviour and the relationship between damage to entities and the Kolmogorov scale of turbulence, sensitivity to fluid dynamic stresses is best studied in turbulent bench scale bioreactors.

There is a growing body of evidence that the ambr™ workstation from TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the physical characteristics of this microscale bioreactor system and found that these are quite different from those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambr™ vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~400 W/m3) when compared to that used at larger scales (typically ~20 W/m3). This specific power input is necessary in order to achieve kLa values sufficiently high to satisfy the oxygen demand of the cells and control of dO2. In line with other studies, we find that the culture of CHO cells in a 15 mL ambr™ bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. Given the differences in physical characteristics between the ambr™ and larger stirred bioreactors, we suggest that this similarity in biological performance is due to their similar control capabilities and the ‘equivalence of the stress parameters’ across the scales when compared with shake flasks.

The commercial production of vanillin from sodium lignosulfonate under highly alkaline conditions, catalyzed by Cu2+ at elevated temperature and pressures up to ∼10 bar, has been simulated in a 3-L stirred reactor. Initially, the process was operated in the presence of nitrogen in dead-end mode, and it was shown that vanillin and vanillic acid were formed by hydrolysis at temperatures of 120, 140, and 160 °C. At the two higher temperatures, the amount of vanillin produced was the same. Subsequently, experiments were conducted at the same elevated pressures and temperatures with addition of air or oxygen-enriched air once the temperature in the reactor had reached temperatures similar to those used when only hydrolysis occurred. In this case, the concentration of vanillin at 140 and 160 °C was equal to that due to hydrolysis, and the subsequent 2-fold increase was due to oxidation. In addition, both vanillic acid and acetovanillone (which has rarely been reported) were produced, as was hydrogen. Thus, for ...

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 105 cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2 ), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h(-1) ) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing.

The effectiveness of a range of impellers for "stirring as foam disruption" (SAFD) is assessed in a vessel of 0.72 m diameter and an aspect ratio of 2: 1. Measurement of power drawn by the impeller achieving SAFD and of the three-dimensional flow field close to the dispersion surface are both used to explain the findings along with the global gas hold-up. A large radial flow Rushton turbine can disrupt foam at a great height but requires high power. Down-pumping hydrofoils are only effective when the ungassed liquid height is below the level of the impeller employed to disrupt foam. Up-pumping hydrofoils are the most effective because their flow pattern gives rise to high velocities across the dispersion surface, which are able to entrain foam in the downflow generated at the walls. © 2002 Elsevier Science B.V. All rights reserved.

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.

Cell-based therapies have the potential to address unmet healthcare needs and improve quality of patient care; effective manufacture is therefore essential. There are, however, many challenges to overcome before this can become a reality and a better understanding of the manufacturing requirements for cell-based products is required. A range of stem cell types are considered as useful cell therapy candidates for various reasons including human embryonic stem cells, induced pluripotent stem cells, and human mesenchymal stem cells (hMSCs). While all have potential as cell-replacement therapies, hMSCs are of particular interest because of their additional immunomodulatory and immune-evasive properties. Until recently, these cells were cultured as a monolayer in tissue culture-treated flasks. However, to produce the number of cells required to meet economically potential demand, it is important to provide greater surface area per unit volume of medium on which the cells can grow. This need has led to the use of microcarriers, particles of some 200. ce:hsp sp=0.25/μm in diameter on which the cells can grow in suspension-based bioreactor systems. After growth, with medium exchange and passaging, the cells have to be harvested. Given the flexibility of stirred tank reactors and the extensive knowledge available from studies of particle suspension within them and their use in free suspension culture, they have become the configuration of choice for culturing adherent cells at increasing scale. This chapter discusses, in detail, the underlying basic principles of stirred tank bioreactors including particle suspension, mass transfer, and the fluid dynamic stresses, which potentially might damage cells. These basic concepts are then related to the cultivation of hMSCs on microcarriers, which has been demonstrated up to the 1000. L scale. It is also shown how concepts from crystallization in stirred tank reactors involving microcarrier-microcarrier and microcarrier-impeller impacts have enabled a successful, insitu harvesting strategy to be developed. The overall concept of growing hMSCs at the minimum agitator speed required for suspension and harvesting them by a short burst of intense agitation in the same bioreactor is introduced. Finally, data are presented showing the success of that approach in 22 combinations involving bioreactors from 15. mL to 5. L with different cell donors, microcarriers (with and without coatings), and detachment enzymes.

The supply of oxygen to a growing organism, aeration, in bioreactors is a critical requirement in biotechnology because of the limited solubility of oxygen in water. Aeration involves transfer of oxygen from the air into the fluid surrounding the organism, from where it is transferred to the organism itself. During the course of a batch bioreaction, oxygen demand often passes through a marked maximum when the species is most biologically active. The basic principles that underlie aeration are exactly the same as those that determine the rate of transfer of any sparingly soluble gas (oxygen) from the gas stream (air) to the unsaturated liquid (broth). In each case, energy is dissipated to increase the rate of transfer beyond simple diffusion, and the mode of energy input defines the two-phase fluid dynamics. Though there are many different types of bioreactor, there are basically four ways of imparting the energy required: rotating agitators (stirred tanks), gas compression (bubble columns/loop fermenters), liquid circulating (jet loop reactors), and shaking (shake flasks, microtiter plates), the last one being common at the screening stage of process development and in clone selection. In addition, the transfer rate depends on the area of contact between the phases, the driving force available (ie, the difference in concentration of oxygen in the two phases), the chemical composition of the liquid, and its temperature. Aeration is used in bacterial, aerobic yeast, polysaccharide, and mycelial fermentations, animal, stem, and plant cell culture, single-cell protein production, and biological aerobic wastewater treatment. Keywords: aeration; bioreactors; mass transfer; oxygen demand; water treatment

The production of vanillin from sodium lignosulfonate under highly alkaline conditions, catalyzed by Cu2+ and at elevated temperature and pressures, has been studied in two sizes of stirred reactor...

Positron emission particle tracking (PEPT) is a relatively new technique allowing the quantitative study of flow phenomena in three dimensions in opaque systems that cannot be studied by optical methods such as particle image velocimetry (PIV) or laser Doppler anemometry (LDA). Here, velocity measurements made using PEPT in two sizes of baffled vessel (∼0.20 m and ∼0.29 m diameter) and two different viscosity fluids agitated by a Rushton turbine are compared for the first time directly in depth with some studies reported in the literature made by LDA for the turbulent regime in the equivalent geometry. Initially, the paper considers how the Lagrangian data obtained by PEPT can be converted into Eulerian in order to make the comparison most effective. It also considers ways of data treatment that improve the accuracy of both the raw PEPT data and the velocities determined from it. It is shown that excellent agreement is found between the PEPT and literature results, especially for the smaller vessel, except for the radial velocity just off the tip of the blade in the plane of the disc of the Rushton turbine. This difference is attributed to the very rapid changes in both magnitude and direction that occurs in that region and also to the different way of ensemble averaging in the two techniques. In addition, the results for the absolute velocities normalised by the impeller tip velocity for all the rectangular cross-section toroidal cells in each size of vessel and each fluid and a range of agitator speeds are compared in the form of frequency histograms. In this analysis, the velocities for each run are obtained from PEPT based on tracking a particle for 30 min and the mean and mode of the velocities each decrease slightly with decreasing scale and Reynolds number. The possible reasons for this variation in the mode and the mean are discussed. Overall, it is concluded that for the radial flow Rushton turbine the PEPT technique can be used to obtain accurate velocity data throughout the entire complex three-dimensional turbulent flow field in an agitated, baffled vessel except very close to the impeller in the radial discharge stream.

Mixing is intrinsically a Lagrangian process and, while Eulerian data are very important, Lagrangian information is necessary for its complete description. Lagrangian data can, in principle, be generated by using numerical simulations or experimental techniques based on Lagrangian tracking to provide the trajectories of fluid elements or particles. We present a set of theoretical and computational tools specifically developed for the analysis and validation of single-phase and multiphase Lagrangian flow data obtained from tracer trajectories in mechanically agitated vessels. The implementation and power of these tools are demonstrated by analyzing a wide range of measurements acquired using the technique of positron emission particle tracking during mixing of Newtonian and non-Newtonian fluids as well as mixing of concentrated solid–liquid suspensions. Finally, the accuracy of the raw Lagrangian data treatment developed here is illustrated by comparing with an Eulerian technique and showing very precise mass continuity for an agitated solid–liquid system. © 2011 American Institute of Chemical Engineers AIChE J, 2012

The literature on micromixing and local-specific energy dissipation rate, (on which it depends), for two-phase systems is limited and conflicting. Here, the competitive iodide/iodate reaction scheme has been used to study the effect of particles and gas flow rate on micromixing in a Rushton turbine agitated vessel. Gassing rates up to 1.5 vvm did not show any effect on product distribution compared to the ungassed at constant mean-specific energy dissipation rate for a feeding near the impeller. Near the upper liquid surface, micromixing improved with increasing flow rate because it increased fluid turbulence. These results confirm the limited literature. With 500 µm glass beads at concentrations up to 2.5 wt.%, micromixing was unaffected near the impeller and near the surface. The related literature is very unclear and it is difficult to draw any precise conclusions. At ∼12 wt.% when cloud formation was observed, micromixing was significantly worse, especially, it is shown for the first time, in the clear layer above the cloud. The latter finding is significant for processes such as precipitation where micromixing determines the particle characteristics.

Micromixing has been studied in a semi-batch reactor agitated by a Rushton turbine using two different reaction schemes. With one scheme, the selectivity was similar at the same mean specific energy dissipation rate and feed location at the 3.6 L scale to that found earlier at the 25 L scale. Also, at the 3.6 L scale, a study of backmixing at different feed locations showed that it was greatest when feeding into the trailing vortex, which is also the best feed location to improve micromixing. At that location, all pipes gave a similar selectivity at the same exit velocity. Comparison with the limited literature showed that one of the two criteria for predicting backmixing fitted the present results quite well but the other was out by two orders of magnitude. The other reaction scheme was used to compare micromixing in a 3 L vessel under baffled conditions with that without them. Such a comparison has only been made once before at a larger 25 L scale and with a different reaction scheme. The earlier surprising result was confirmed, namely that the mean specific energy dissipation rate required for equivalent selectivity without baffles was less than that required with them.

The three-component flow field and spatial phase distribution of binary mixtures of glass particles suspended in water in a stirred vessel have been resolved using positron emission particle tracking (PEPT). The Lagrangian flow data provided by the technique have been converted to give a detailed Eulerian description of each of the three phase components of the flow generated by a pitched blade turbine. For the first time, it has been possible to determine the full 3D velocity and concentration fields of the liquid phase and both the solid components within opaque dense slurries of this type containing up to 40 wt % solids. Spatial distributions of local time-averaged slip velocity have also been obtained for each solid component showing wide variations. The detailed PEPT measurements have enabled the solids mass balance and the mass continuity of the three phase components to be accurately verified throughout the vessel. © 2010 American Institute of Chemical Engineers AIChE J, 2011

If 100 dice cannot be cast simultaneously, one single die can be cast 100 times. On the basis of this simple principle, the experimental technique of positron emission particle tracking has been used to develop and implement a new methodology for quantifying the local and global mixing characteristics within a mechanically agitated fluid batch system. This Lagrangian technique uses a single positron-emitting particle as flow follower. Using a high data acquisition rate, such a tracer is continuously tracked in 3D space and time to accurately determine its trajectory over a considerable period of time. By partitioning its long trajectory, the single particle tracer can be regarded as thousands of simultaneously tracked particles which are instantaneously, locally and non-invasively injected in the mixing system at varying feed positions. A large amount of PEPT data were collected for impeller rotational speeds ranging from 100 to 500 rpm which allowed new statistical tools derived from information theory, such as Shannon entropy and uncertainty, to be implemented in the data analysis. Thus, measurements of entropy mixing indices were obtained as a function of position, time and impeller speed. The method also allowed the determination of characteristic time parameters including the macroscale mixing time which agreed very well with correlations of the dimensionless mixing time available in the mixing literature. Detailed local information is provided on minimum mixing time positions for feed and withdrawal of material, which can be used to optimise the design or operation of stirred batch mixing systems.

Two-dimensional particle image velocimetry (PIV) is usually used to determine the complex flow field in mechanically agitated vessels on the basis of measurements taken in a single vertical plane, thus, assuming axial symmetry. In this paper, we use 2D PIV to investigate the effects of the azimuthal position of the measurement plane in a fully baffled vessel agitated by a pitched blade turbine. Seventeen planes located at 5 degree intervals between two adjacent baffles are analysed. To maintain a high spatial resolution of ∼1 mm when examining each plane, a two-block approach is employed combining data from two fields of view to reconstruct the whole flow field. Time-averaged velocity and turbulent kinetic energy fields are obtained under fully turbulent conditions as a function of the azimuthal position of the laser plane. It is shown that the assumption of axial symmetry for such Eulerian fields is not fully justified within a fully baffled vessel, as there are considerable differences between planes. The results also show for the type and size of impeller used here, the importance of including both the axial and radial discharge contributions for an accurate evaluation of the flow number, otherwise it can be underestimated by up to 60%. The three-dimensional nature of the PIV measurements has also enabled the mass continuity to be accurately verified throughout the vessel.

The technique of positron emission particle tracking (PEPT) has been used to determine the two-phase flow field and spatial phase distribution in a stirred vessel containing coarse glass particles suspended in water. The Lagrangian flow data provided by PEPT have been converted to give a detailed Eulerian description of the two-phase flow generated by a pitched blade turbine (PBT) operating in up-pumping or down-pumping mode. For the first time, it has been possible to determine the full 3D velocity and concentration fields of both the liquid and the solid phase within an opaque dense slurry of this type containing up to 40 wt% solids. The detailed PEPT measurements have also enabled the solids mass balance and the mass continuity of the two phases to be accurately verified throughout the vessel. The data show that the pumping effectiveness of the PBT is unaffected at moderate solid fractions, but is considerably reduced at high fractions and even more so in the up-pumping mode. A uniformity index based on the variance of the local solids concentration in the vessel shows that, overall, a down-pumping PBT achieves a significantly better homogeneity than an up-pumping PBT.

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO(2) evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer's yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO(2) evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing.

The agitator speed required to suspend solids under gassed conditions, N JSg , has been studied in water and in corn syrup of 0.01 and 0.1 Pas giving Reynolds numbers from the full turbulent region down to ∼103. Of the impellers tested, the down-pumping, three-blade, axial flow hydrofoil impellers are generally unsuitable for this duty, and although six-blade, mixed flow down-pumping impellers require the lowest mean specific energy dissipation rates to suspend the solids, (ϵ T ) JSg , at low gas flow rates, they are still prone to flow instabilities and torque fluctuations. The latter poor characteristics are made worse by reducing the size of the impeller relative to the vessel and by increasing viscosity and gas flow rate, Q GV . Thus, they are of limited use for such systems. The Ekato InterMIG impeller has the highest (ϵ T ) JSg and tends to cause vessel vibrations when dispersing the gas, and this weakness is also enhanced by increasing viscosity and gas flow rate. Again, they are generally not appr...

Positron emission particle tracking (PEPT) is a new technique allowing the quantitative study of flow phenomena in three dimensions in opaque systems that cannot be studied by techniques based on optical methods such as particle image velocimetry (PIV) or laser Doppler anemometry (LDA). It has previously been used for studying solid particle motion in various systems used in particulate processing. Here, for the first time, velocity measurements made using PEPT with a down-pumping pitched blade turbine (PBTD) are compared directly with those made by PIV in water in the same equipment. It is shown that excellent agreement is found between the two methods except just below the impeller in the discharge. However, this difference is attributed to the different type of data collected and the different way of ensemble-averaging in the two techniques. Similar results were found at higher agitator speeds with both the PBTD and an up-pumping PBT (PBTU) where a small amount of surface aeration occurred. Measurements in solid liquid systems with surface aeration at 0.5 wt% solids or higher were not possible with PIV, but excellent results were obtained with PEPT for both the PBTD and PBTU in a 5 wt% suspension. It is concluded that this calibration study shows that the PEPT technique can be used to obtain accurate velocity data throughout all of the complex three-dimensional flow field in a range of mechanically agitated, turbulent, multi-phase systems previously not amenable to quantitative analysis.

The technique of positron emission particle tracking (PEPT) is a unique non-intrusive Lagrangian flow visualisation technique which allows probing of opaque fluids and within opaque apparatus. It uses a single positron-emitting particle as flow tracer which is tracked in 3D space and time to reveal its full Lagrangian trajectory. PEPT was used to study the mixing of a concentrated suspension of coarse glass particles, containing up to 10.6 wt% solids, in a vessel agitated by a pitched blade turbine operating in both up-pumping and down-pumping modes. The Lagrangian information obtained was used to obtain a detailed Eulerian description of the two-phase flow inside the vessel. For the first time, it has been possible to determine the full 3D velocity and concentration fields of both the liquid and the solid phase within an opaque flow of this type.

The gas flow in a 3:1 aspect ratio vessel agitated by triple Rushton turbines has been measured by an ultrasound Doppler probe and by means of residence time studies. Strong recirculation around each impeller is found which fits in well with the compartmentalisation found in earlier liquid mixing studies. Surprisingly, when two axial A315 impellers above a Rushton turbine were used, gas recirculation around each impeller was still found. Study of the liquid phase mixing by a decolourisation technique confirmed that the gas flow essentially destroyed the strong axial liquid flow expected. Indeed, even under unaerated conditions, compartmentalisation was found between each impeller. On a mesure a l'aide d'une sonde Doppler a ultrasons et par des etudes du temps de sejour l'ecoulement gazeux dans un reservoir (rapport des dimensions caracteristiques de 3:1) agite par trois turbines Rushton. On a observe une forte recirculation autour de chaque turbine que correspond bien a la compartimentalisation trouvee dans les etudes de melange liquide precedentes. Etonnamment, lorsqu'on utilise deux turbines axiales A315 au-dessus d'une turbine Rushton, on constate encore une recirculation du gaz autour de chaque turbine. L'etude du melange de la phase liquide par une technique de decoloration confirme que l'ecoulement gazeux detruit essentiellement le fort debit axial attendu. En effet, meme dans des conditions non aerees, une compartimentalisation est observee entre chaque turbine.

The hydrodynamics of a model bioreactor stirred by an ‘Elephant Ears’ impeller, either up- (EEU) or down-pumping (EED) have been studied under unaerated and aerated conditions. For the latter, air flow rates of 0.01–0.05 vvm (typical for animal cell culture) and 0.5 vvm (at the lower end of those used for bacterial fermentations) were used. For all these four conditions, the detailed flow of the liquid phase was studied using particle image velocimetry (PIV) with red fluorescent seeding particles, which emitted at a different frequency from the laser light. This approach enabled exclusion of the laser light scattered by the bubbles using an optical filter. From these measurements, quantitative global flow fields, axial velocity profiles, flow numbers, turbulent kinetic energy and vorticity have been determined. In addition, power, power numbers and hold-up were obtained. For the up-pumping mode, turbulent power numbers were not affected at any aeration rate, i.e., P g / P ≈ 1 whilst for the down-pumping EE impeller, P g / P decreased by up to 30% at the highest rate. In addition, the hold-up was greater in the up-pumping mode. The global flow field and mean liquid velocities were not significantly affected at the lower air flow rates, but for the highest aeration rate of 0.5 vvm, the liquid velocities were decreased regardless of pumping mode. Maximum values of turbulent kinetic energy (TKE) in the discharge stream were relatively unaltered, but the distribution of TKE in the bulk changed significantly with the mean values decreasing with increasing aeration. Flow numbers decrease by 20–30% from the unaerated case at the highest air flow rate, especially for the EED. The results show that, as with other axial flow impellers, the up-pumping mode offers significant advantages over the down flow.

Up-pumping pitched blade turbines (and similar impellers) have recently been shown to be particularly effective for achieving a variety of mixing duties. Here, their turbulent flow characteristics are analysed by angle-resolved particle image velocimetry (PIV) for the first time and compared with their down-pumping equivalent, the usual time-averaged parameters also being determined for each. The work was conducted in 0.15 m diameter vessel ( T ) with a 45° impeller of diameter D (=0.45 T ) in water. The angle-resolved PIV enables a number of novel features to be identified. Firstly, the two pumping directions are shown to give very different vortex structures, even though the flow numbers, Fl, are the same (=0.79). In addition, the ‘spottiness’ of the normalized kinetic energy along a radius as the trailing vortex moved away from each impeller can be identified, which is not shown from time-averaged data. Often, the most important parameter for processing is the local normalized specific energy dissipation rate, e T * and this is estimated using three methodologies: by measurement of the components of the stress tensor directly, ( e T * ) DE ; by dimensional analysis, ( e T * ) DA , with measured integral length scales (ILS); and by the Smagorinsky closure method, ( e T * ) SGS , to model unresolved scales (with a Smagorinsky constant used in the literature on stirred vessels). Again, only the angle-resolved results show the spottiness of e T max * and also higher values than the time-averaged. Differences in the values obtained by the three methods are discussed and compared with the existing literature. Most importantly, for the first time, the power input in the PIV-interrogated region is calculated from the three methods and compared to the input based on the impeller torque. Both DA and SGS methods are shown to overestimate the true power by a factor of 5 and 2, respectively, whilst the DE method provided a significant underestimate (1/5th) due to the limitation of the resolved length scales. The SGS method shows the greatest promise and by changing the value of the Smagorinsky constant in accordance with recent recommendations, good agreement is obtained. Nevertheless, it is concluded that there is still a need for improved methods for determining the important mixing parameter, e T max * .

This paper considers recent work, mainly from the University of Birmingham, on stirred bench scale bioreactors. It focuses on mixing as it affects biological performance on scale-up, particularly the impact of stress on organisms as a result of fluid dynamic phenomena (mixing), namely mechanical stresses or spatial and temporal inhomogeneities. The work uses new experimental techniques (flow cytometry and advanced image analysis) and covers mycelial, bacterial (both including GMO’s) and yeast (including beer) fermentations and animal cell culture in batch, fed-batch and chemostat conditions. By using gas blending to control dissolved oxygen (dO2), the impact of mechanical stresses due to agitation and aeration can be decoupled from dO2 effects. With mycelia, image analysis of all biomass shows damage from mechanical stresses generated by agitation but not by aeration; and that productivity may be affected. Also, on scale-up, such damage is reduced. With yeast and bacteria, flow cytometry does not indicate damage. On the other hand, plant scale studies with E. coli give lower biomass but higher cell viability compared to the well-mixed bench-scale. When the locally high nutrient, low dO2 and high pH values found on the plant scale near feed points are simulated at bench-scale, similar results to plant scale are obtained. Similar scale-down results are obtained with a recombinant E. coli. Simulated large-scale beer production, where the inhomogeneities are associated with yeast distribution and temperature, also indicates a poorer performance. Though perceived to be more sensitive to mechanical stresses, somewhat similar conclusions can be drawn for animal cell culture, with stresses associated with bursting bubbles being potentially the most significant. These perceptions lead to problems associated with high dissolved carbon dioxide (pCO2) and osmolality in large-scale cell culture. A related topic is the search for ‘low shear’ impellers, a concept useful to mixer manufacturers but misleading to users.