Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus.

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.

Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: lessons learnt and what the future holds.

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.

Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus.

Insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. Initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Spodoptera frugiperda (Sf9) cells. The percentage of cells infected with wild and recombinant baculovirus AcNPV and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were in when infected and by the multiplicity of infection (MOI). However, the cell density at the time of infection and the medium condition showed little direct influence on infectivity. There may, however, be an indirect influence in that these factors determine the relative distribution of cells in the cell cycle. The infectivity is then in turn affected by the relative frequency of cells in the G1, S and G2/M phases. Insect cell specific oxygen uptake rates (1.3-3.4 x 10(-17) mol per cell per s) were essentially similar to or less than those measured for hybridoma cells. However, when Sf9 cells were infected with baculovirus, the specific oxygen uptake rate increased by up to 40%.

Further studies of the culture of mouse hybridomas in an agitated bioreactor with and without continuous sparging.

TB/C3 mouse hybridoma cells have been grown at 2 controlled dO2 conditions by headspace and sparged oxygenation. Also a variety of sparging rates and sparger sizes and positions have been employed. Headspace oxygenation at dO2 levels from 5% to 100% of saturation give essentially the same performance as controls. Sparging is generally damaging to cells, the extent of damage decreasing with reduced sparging rate until at below about 0.02 vvm results equivalent to the unsparged conditions are obtained. Damage is clearly linked with bubble-cell interactions at the air-medium interface where bubbles bursting in clusters and of a size less than 5 mm appear to be the most lethal. When the interaction of air sparging with the agitator flow leads to an increase in the number of smaller bubbles and cluster bursts, cell damage is further increased. Pluronic F-68 reduces damage very significantly. Biological aspects are briefly discussed in the light of various biological tests. The practical implications of this work for large scale, free suspension cell culture are outlined.

The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations.

Multi-parameter flow cytometric techniques have been developed for the 'at-line' study of bacterial cultivations. Using a mixture of specific fluorescent stains it is possible to resolve an individual cells physiological state beyond culturability, based on the presence or absence of an intact polarised cytoplasmic membrane, enabling assessment of population heterogeneity. It has been shown that during the latter stages of small-scale (5 l), well mixed fed-batch cultivations there is a considerable drop in cell viability, about 17%, as characterised by cytoplasmic membrane depolarisation and permeability. These phenomena are thought to be due to the severe and steadily increasing stress associated with glucose limitation at high cell densities, during the fed-batch process. Such effects were not found in either batch or continuous culture cultivations. The possibility of using these findings for improved process control using 'on-line' flow cytometry are discussed.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Break-up, coalescence and catastrophic phase inversion in turbulent contactors.

When a low concentration of immiscible phase is dispersed, break-up in the impeller region controls the drop size. The traditional application of Kolmogoroff's theory of local isotropic turbulence has been moderately successful in relating equilibrium drop sizes to the physical properties and the turbulent flow, with low power number impellers giving smaller drops at equal mean specific energy dissipation rates, [Formula: see text] However, to explain the reduction in drop size at equal [Formula: see text] on scale-up, the concept of intermittency must be introduced leading to a scale-up rule close to constant tip speed. With increasing concentration, coalescence generally becomes important and drop sizes increase. Modelling of coalescence involves collision frequency and coalescence efficiency. The latter is dependent on the type of drop interface, the establishment of which type for a particular system being difficult. The difficulty is compounded since in clean systems, at concentrations of the aqueous phase > approximately 20% by volume, droplets of oil appear in the aqueous drops whilst the converse is not found. At sufficiently high concentrations, where the concept of collision frequency is questionable, catastrophic phase inversion (CPI) occurs because coalescence becomes so high. Anything that enhances coalescence, e.g. surfactants, particles that bridge interfaces, wettable surfaces, bulk flow patterns, encourages CPI to occur at lower concentrations of dispersed phase. Satisfactory models for CPI are not available.

Oxygen transfer in animal cell culture medium.

Both k(L)a and k(L) measurements were carried out by an unsteady state technique at impeller speeds ranging from 1.6 to 5.8 s(-1) in a mechanically agitated animal cell culture vessel of working volume 1.5 L. Checks were made that the time constant of the oxygen electrode was negligible compared to the time for aeration and that the oxygen electrode reading was not a function of agitator speed in the range employed. The k(L) values by surface aeration of (1.18-3.54) x 10(-5) m/s and k(L)a values by sparged aeration of (2.8-8.5) x 10(-4) s(-1) were found. The former are in reasonable agreement with published experimental values and the latter in accord with values estimated from published correlations based on agitator power input and aeration rate. The fluids used were water, basal medium, and basal medium supplemented with 5% (v/v) foetal calf serum; for each of these, k(L) and k(L)a values were similar. However, the addition of silicone antifoam (6 PPM) reduced the k(L)a value by ca. 50%.

Control of pH in large-scale, free suspension animal cell bioreactors: alkali addition and pH excursions.

It is likely that, in the future, animal cell cultures of a higher cell density and/or cell lines with higher specific oxygen demands will be available. Such developments will lead to the need for improved homogeneity in the bioreactor and a greater supply of nutrients. The accompanying significant increase in CO(2) production and accumulation and the resulting reduction in pH are also important implications for process engineering. Such pH reduction is typically controlled by the addition of sodium carbonate. Previous studies using flow visualization mimicking the operating conditions in a typical plant-scale reactor showed potentially cell-damaging regions within it due to pH excursions. This paper confirms the existence of these excursions by pH measurements in the alkali addition zone. It also identifies the accumulation of alkali in a region of poor local liquid homogenization as a serious scale-up problem and shows how a change in the addition point significantly reduces it.

Gas-liquid dispersion with dual Rushton turbine impellers.

Aerated and unaerated power consumption and flow patterns in a 0.56 m diameter agitated vessel containing water with dual Rushton turbines have been studied. Under unaerated conditions with a liquid height-to-diameter ratio of 2, an impeller spacing of 2 to 3 times the impeller is required for each to draw an amount of power equal to a single impeller. For aerated conditions, if a similar spacing is used, equations for the flooding-loading transition and for power consumption for a single Rushton impeller can be extended relatively easily to dual systems. All results for this spacing are explained by reference to bulk flow patterns and gassed-filled cavity structures and the proportion of sparged gas flowing through the upper impeller is also estimated. Such a spacing is generally recommended since it maximizes the power draw and hence the potential for oxygen mass transfer. Data are presented for other spacings but the results do not fit in easily with single agitator studies because strong impeller-impeller flow pattern interactions occur.

A fluid dynamic study of the retrofitting of large agitated bioreactors: Turbulent flow.

Studies were conducted in three 19-m(3) fermentors (14 m(3) working volume, aspect ratio = 3:1), one fitted with four Rushton turbines (D/T = 0.35), one with three Lightnin' A315 hydrofoil impellers (D/T = 0.46). The power drawn under the same aerated conditions relative to the unaerated ones was always greater with the hydrofoils, which gives them the potential for enhanced mass transfer rates under practical operating conditions. However, the power draw was also sensitive to the magnitude of the unaerated power. Indeed, at low unaerated specific power ( approximately 0.6 W-kg) and high air flow rates ( approximately 1vvm), the relative power draw with the hydrofoils could be even greater than 1. The hold-up with each of the impellers was broadly similar at the same aeration rate and power input, though the later had a much smaller impact in these large vessels than has been reported in the literature based on smaller scale work. As usual, repressed coalescence caused increased hold-up, and, with the hydrofoils, this increase was associated with a lower power draw. Because of the greater mechanical vibration of the reactors with the hydrofoils, vibration characteristics of the vessels were measured and they were very similar. The results showed that provided care is taken in the mechanical design of the system, such impellers can operate reliably in large-scale fermentations with the potential for enhanced biological performance. (c) 1994 John Wiley & Sons, Inc.

Action of shear on enzymes: studies with alcohol dehydrogenase.

Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C.

Oil/water and pre-emulsified oil/water (PIT) dispersions in a stirred vessel: Implications for fermentations.

This study examines dispersions of rapeseed oil (RSO) in water by mechanical agitation under conditions mimicking those found in certain antibiotic fermentations; for example, in the presence of air, antifoam, and finely divided CaCO(3) particles. A problem with residual oil has been reported for such fermentations, and it has been suggested that the use of pre-emulsified oil can reduce this problem. Hence, the dispersion of a pre-emulsified oil produced by the "phase inversion temperature (PIT) method" has been evaluated. In both cases, the volume fraction of oil was 2%. For the RSO systems, a relatively high agitation speed was required to disperse the oil, especially in the presence of the particles and, when the agitation was stopped, separation occurred rapidly. The Sauter mean drop diameters depended on the system, being at an average energy dissipation rate of approximately 0.9 W kg(-1), 180 microm for RSO/water, 130 microm for RSO/water(antifoam)/air, 580 microm for RSO/water/CaCO(3), and 850 microm for RSO/water(antifoam)/air/CaCO(3). For the same four systems, the PIT emulsion, once dispersed, was very stable and the drop size was essentially independent of the operating conditions, with a Sauter mean diameter of approximately 0.3 microm. The implications of these findings for fermentations in which oil is used as a carbon source are assessed.

A fluid dynamic study using a simulated viscous, shear thinning broth of the retrofitting of large agitated bioreactors.

Studies were conducted(1) in 19-m(3) fermentors (14-m(3) working volume) using four Rushton turbines, four Prochem Maxflo Ts, and three Lightnin' A315s and the results in water have been reported earlier. Here, a 1.7 wt/vol% Xanthan solution has been used as the working fluid, simulating viscous broths to give Reynolds numbers (Re) between 1800 and 4500. As predicted from small-scale studies, the power numbers at these values of Re were similar to those in water. The K factor (the ratio of power draw under aerated conditions compared to non-aerated) was the same as in water at the higher values of Re, but at the lower values it fell more rapidly with increasing aeration rate and to a lower value than in water. At all times, K was higher than with Rushton turbines. Vibration characteristics were also measured. Under aerated conditions, the fermentors vibrated with an amplitude 75% to 100% less than in water due to viscous damping. With increasing air flow, the amplitude increased steadily due to the presence of very large and rapidly rising bubbles in such fluids to give values 2.5 to 3 times those in water. Nevertheless, these mechanical problems can be overcome, allowing such agitators to be used successfully in high viscosity mycelial fermentations.

Study of drop and bubble sizes in a simulated mycelial fermentation broth of up to four phases.

The mean sizes and size distributions of air bubbles and viscous castor oil drops were studied in a salt-rich aqueous solution (medium), first separately, and then simultaneously as a three-phase system. The dispersion was created in a 150-mm-diameter stirred tank equipped with a Rushton turbine, and the sizes were measured using an advanced video technique. Trichoderma harzianum biomass was added in some experiments to study the effect of a solid phase under unaerated and aerated conditions to give either three-or four-phase systems. In all cases, the different dispersed phases could be clearly seen. Such photoimages have never been obtained previously. For the three phases, air-oil-medium, aeration caused a drastic increase in Sauter mean drop diameter, which was greater than could be accounted for by the reduction in energy dissipation on aeration. Also, as in the unaerated case, larger drops were observed as the oil content increased. On the other hand, mean bubble sizes were significantly reduced with increasing oil phase up to 15% with bubbles inside many of the viscous drops. With the introduction of fungal biomass of increasing concentration (0.5 to 5 g L(-1)) under unaerated conditions, the Sauter mean drop diameter decreased. Finally, in the four-phase system (oil [10%]-medium-air-biomass) as found in many fermentations, all the phases (plus bubbles in drops) could clearly be seen and, as the biomass increased, a decrease in both the bubble and the drop mean diameters was found. The reduction in size of bubbles (and therefore increase in interfacial area) as the oil and bio- mass concentration increased provides a possible explanation as to why the addition of an oil phase has been reported to enhance oxygen transfer during many fermentations.

Improved performance in viscous mycelial fermentations by agitator retrofitting.

For viscous mycelial fermentations it was demonstrated at the pilot-plant scale that the replacement of standard radial flow Rushton turbines with larger diameter axial-flow Prochem hydrofoil impellers significantly improved oxygen transfer efficiency. It was also determined that the Streptomyces broth under evaluation is highly shear thinning. Separate experiments using a Norcardia broth with similar Theological properties demonstrated that the oxygen transfer coefficient, K(L)a, can be greatly increased by use of water additions to reduce broth viscosity. These observations are consistent with the hypothesis that the improvement in oxygen transfer by changing agitator types is primarily due to an improvement in bulk mixing. A model is presented, based on the concepts of Bajpai and Reuss, which explains this improvement in performance in terms of enlargement of the well mixed micromixer region for viscous mycelial broths.

Dependence of mycelial morphology on impeller type and agitation intensity.

The influence of the agitation conditions on the morphology of Penicillium chrysogenum (freely dispersed and aggregated forms) was examined using radial (Rushton turbines and paddles), axial (pitched blades, propeller, and Prochem Maxflow T), and counterflow impellers (Intermig). Culture broth was taken from a continuous fermentation at steady state and was agitated for 30 min in an ungassed vessel of 1.4-L working volume. The power inputs per unit volume of liquid in the tank, P/V(L), ranged from 0.6 to 6 kW/m(3). Image analysis was used to measure mycelial morphology. To characterize the intensity of the damage caused by different impellers, the mean total hyphal length (freely dispersed form) and the mean projected area (all dispersed types, i.e., also including aggregates) were used. [In this study, breakage of aggregates was taken into account quantitatively for the first time.]At 1.4-L scale and a given P/V(L), changes in the morphology depended significantly on the impeller geometry. However, the morphological data (obtained with different geometries and various P/V(L)) could be correlated on the basis of equal tip speed and two other, less simple, mixing parameters. One is based on the specific energy dissipation rate in the impeller region, which is simply related to P/V(L) and particular impeller geometrical parameters. The other which is developed in this study is based on a combination of the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. For convenience, the function arising from this concept is called the "energy dissipation/circulation" function.To test the broader validity of these correlations, scale-up experiments were carried out in mixing tanks of 1.4, 20, and 180 L using a Rushton turbine and broth from a fed-batch fermentation. The energy dissipation/circulation function was a reasonable correlating parameter for hyphal damage over this range of scales, whereas tip speed, P/V(L), and specific energy dissipation rate in the impeller region were poor. Two forms of the energy dissipation/circulation function were considered, one of which additionally allowed for the numbers of vortices behind the blades of each impeller type. Although both forms were successful at correlating the data for the standard impeller designs considered here, there was preliminary evidence that allowing for the vortices would be valuable. (c) 1996 John Wiley & Sons, Inc.

The use of flow cytometry to study the impact of fluid mechanical stress on Escherichia coli W3110 during continuous cultivation in an agitated bioreactor.

Continuous culture fermentations of Escherichia coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also the cell size did not change, results confirmed by SEM and TEM. There were no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles.

The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products.

Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained. These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification.

Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency.