Growth models of cultures with two liquid phases. 8. Experimental observations on droplet size and interfacial area

Because of the importance of the drop she distribution and interfacial area of the dispersed liquid phase in hydrocarbon fermentations, experiments were carried out to determine the drop size distribution and the interfacial area during batch fermentations of Candida lipolytica on gas oil and on n-hexadecane dissolved in dewaxed gas oil. The effects of cell concentration and dispersed phase volume fraction on size distribution and interfacial area were investigated. Measurements of interfacial tensions, densities, viscosities, and fatty acid concentrations were also made. The results show that the size distribution is skewed and that the Sauter mean diameter is in the range of 10 to 30 μ. Both the Sauter mean diameter and the interfacial area increased during the course of a batch fermentation; however, they decreased at the end of the fermentation. The interfacial area also increased with inoculum size.     

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.     

Stochastic modelling of axial dispersion in external-loop airlift bioreactors

The paper presents a model of the motion of a particle subjected to several transport processes in connection with mixing in two phase flow. A residence time distribution technique coupled with a one-dimensional dispersion model was used to obtain the axial dispersion coefficient in the liquid phase, D_ax. The proposed model of D_ax for an external-loop airlift bioreactor is based on the stochastic analysis of the two-phase flow in a cocurrent bubble column and modified for the specific flow in the airlift reactor. The model takes into account the riser gas superficial velocity, the riser liquid superficial velocity, the Sauter bubble diameter, the riser gas hold-up, the downcomer-to-riser cross sectional area ratio. The proposed model can be applied with an average error of ᆨ.     

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.     

Bubbling and foaming assisted clearing of mucin plugs in microfluidic Y-junctions

Microfluidic Y-junctions were used to study mechanical mechanisms involved in pig gastric mucin (PGM) plug removal from within one of two bifurcation branches with 2-phase air and liquid flow. Water control experiments showed moderate plug removal due to shear from vortex formation in the blockage branch and suggest a PGM yield stress of 35 Pa, as determined by computational fluid dynamics. Addition of hexadecyltrimethylammonium bromide (CTAB) surfactant improved clearing effectiveness due to bubbling in 1 mm diameter channels and foaming in 500 μm diameter channels. Plug removal mechanisms have been identified as vortex shear, bubble scouring, and then foam scouring as air flow rate is increased with constant liquid flow. The onset of bubbling and foaming is attributed to a flow regime transition from slug to slug-annular. Flow rates explored for 1 mm channels are typically experienced by bronchioles in generations 8 and 9 of lungs. Results have implications on treatment of cystic fibrosis and other lung diseases.     

Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.     

Substrate controlled development of anaerobic acidifying aggregates at different shear rates in a gas lift reactor.

The influence of liquid shear rates on the development of acidifying mixed-culture aggregates was studied in a gas-lift reactor. The glucose concentration was kept at a constant and relatively high level by operating the reactor in pH-auxostat mode. Size, strength, and wet density of aggregates cultivated at different superficial gas velocities (Ug) were investigated. Image analysis showed that the Sauter mean diameter (Ds) decreased with increasing Ug. A stirred tank was used to characterize the surface detachment rate (Rd) under non-growth conditions. An exponential decrease was observed in Rd with the applied Ug during cultivation, i.e., aggregates became stronger. The increased strength coincided with an increase in aggregate wet density. Size classified aggregates showed an increase in Rd with the square of the aggregate diameter (Dp), however, this contribution was much smaller than the effect of adaptation. Experiments in a similar gas-lift reactor under dynamic conditions without adaptation, showed that Rd increased exponentially with increasing Ug. So, two important contributions to Rd can be distinguished: adaptation, which induces stronger aggregates, and aggregate size, which makes them less susceptible to hydrodynamic shear. A general expression for Rd was derived, which depends on Dp and Ug. Combining this equation with the surface biomass growth rate (Rg) allowed for the estimation of the maximal diameter (Dmax) aggregates can reach at any Ug, and it was found that the estimated and measured Dmax were in good agreement.     

Mechanical stress tolerance of two microalgae

Aim of the present work is quantifying the mechanical stress generated by some major process equipment used in massive microalgae culturing plants (centrifugal and air-lift pumps, and nozzles) and highlighting its effects on the microalgal population. Two microalgal species were used as test cases: Chlorella vulgaris (unicellular) and Scenedesmus dimorphus 1237 (colonial). The evaluation of the shear effect on algal growth was carried out through measurement of absorbance, photosynthetic activity (oxygen evolution) and variable chlorophyll fluorescence. Cell aggregate development/breakage was effectuated by visual inspection and light scattering. The use of centrifugal pumps for culture recycling strongly affected the growth of C. vulgaris, while nozzles effects were confined to aggregate breakage of S. dimorphus. The analysis of experimental data is supported by the consideration of hydrodynamic stress calculated by: shear rate, shear stress, stress volumes/times, energy dissipation rates, and turbulence microscale size.     

Analysis of particle strain in stirred bioreactors by drop breakage investigations

Understanding of particle strain and drop breakage is relevant for various technical applications. To analyze it, single drop experiments in a breakage cell and evolving drop size distributions in an agitated system are studied. The mechanisms for particle strain and drop breakage are assumed to be comparable for the investigated turbulent flow regime. The agitation process is simulated using a population balance model. This model provides transient prediction capacities at different scales and can be used for scale-up/down projects. The number and the size distributions of daughter fragments for single drops have been studied. The results clearly support the assumption of binary breakage. The most common assumption of a Gaussian distribution for the daughter drop size distribution could not be supported. The evolution of a breakage-dominated toluene/water system was then simulated using different daughter drop size distributions from literature. The computational results were compared with experimental values. All simulations were able to predict the transient Sauter mean diameter excellently but varied strongly in the results on the shape of the distribution. In agreement with the experimental single drop results, the use of a bimodal or a very broad bell-shaped distribution of the daughter drops is proposed for the simulations. Although these results were obtained in a particular vessel for a specific phase system, it can be applied to simulate transient multiphase systems at different scales. We would expect that the general trends observed in this study are comparable to various applications in multiphase bioreactors.     

Pressure drop and flow rate measurements in a human aortic bifurcation cast for steady and pulsatile flow

Pressure drop and flow rate measurements in a rigid cast of a human aortic bifurcation under both steady and physiological pulsatile flow conditions are reported. Integral momentum and mechanical energy balances are used to calculate impedance, spatially averaged wall shear stress and viscous dissipation rate from the data. In the daughter branches, steady flow impedance is within 30% of the Poiseuille flow prediction, while pulsatile flow impedance is within a factor of 2 of fully developed, oscillatory, straight tube flow theory (Womersley theory). Estimates of wall shear stress are in accord with measurements obtained from velocity profiles. Mean pressure drop and viscous dissipation rate are elevated in pulsatile flow relative to steady flow at the mean flow rate, and the exponents of their Reynolds number dependence are in accord with available theory.     

Dispersions in hydrocarbon fermentation. A retrospective study

A nondimensionalized plot, obtained by normalizing the drop-size distribution in the hydrocarbon phase using the Sauter mean diameter, shows a tendency towards self-preservation of the distribution. Changes of distribution in time during the course of fermentation, initial dispersed phase fraction, speed of rotation, and reactor size were taken into account. Using this self-preserving property, an empirical (single parameter) equation has been proposed for drop-size distribution. Data, available from the literature, are presented for non-biological and biological systems (gas-oil, n-hexadecane, and n-hexadecane dissolved in dewaxed gas oil as dispersed phases). The parameter, Sauter mean diameter, has been correlated with the operating conditions, and a critical review presented. Cell density was found to have significant effect on Sauter mean diameter. This effect has also been empirically explained. The possibilities of using generalized distribution in predicting the performance of fermenters is outlined.     

Isolation and purification of serum and interfacial peptides of a trypsinolyzed β-lactoglobulin oil-in-water emulsion

Information on the conformation of proteins adsorbed to an oil–water interface is usually determined by following the time course of enzymatic hydrolysis of the protein in an oil-in-water emulsion. Unlike previous works reported in the literature, the research presented in this paper provides information on which peptides are actually in contact with the lipid bilayer (interfacial peptides) and those segments that project into the aqueous phase (serum peptides). In order to achieve this classification of peptides, we present a method to separate serum peptides from interfacial peptides by initial centrifugation steps followed by reversed-phase high-performance liquid chromatography. The effectiveness of the method was ascertained by performing proteolysis on β-lactoglobulin adsorbed to an oil-water interface in a soybean oil-water emulsion. It was found that more peptides are qualitatively and quantitatively found adsorbed to the oil–water interface as compared to peptides released into the serum.     

Characteristics of hydrocarbon uptake in cultures with two liquid phases

In hydrocarbon fermentation, the efficiency of hydrocarbon uptake by cells ins one of the keys to the economical production of single-cell protein. This work is concerned with characterization of cultures with two liquid phases for understanding the hydrocarbon uptake process by cells. Batch cultivation of Candida lipolytica was carried out in shaking flasks and in a tower fermentor with motionless mixers. Micorscopic observation and cell and hydrocarbon concentration distribution in batch cultivation showed that some cells are attached to the large oil drops ad others are free from them. Interfacial tension between oil and water and Sauter mean drop size decreased as cultivation proceeded. On the basis of the experimental results, the process of hydrocarbon uptake by cells is discussed.     

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.     

Rheology and ultrasound scattering from aggregated red cell suspensions in shear flow

The shear flow dynamics of reversible red cell aggregates in dense suspensions were investigated by ultrasound scattering, to study the shear disruption processes of Rayleigh clusters and examine the effective mean field approximation used in microrheological models. In a first section, a rheo-acoustical model, in the Rayleigh scattering regime, is proposed to describe the shear stress dependence of the low frequency scattered power in relation to structural parameters. The fractal scattering regime characterizing the anisotropic scattering from flocs of size larger than the ultrasound wavelength is further discussed. In the second section, we report flow-dependent changes in the low-frequency scattering coefficient in a plane-plane flow geometry to analyze the shear disruption processes of hardened or deformable red cell aggregates in neutral dextran polymer solution. Rheo-acoustical experiments are examined on the basis of the rheo-acoustical model and the effective medium approximation. The ability of ultrasound scattering technique to determine the critical disaggregation shear stress and to give quantitative information on particle surface adhesive energy is analyzed. Lastly, the shear-thinning behavior of weakly aggregated hardened or deformable red cells is described.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Hydraulic requirements of stream communities: a case study on invertebrates

1. We relate invertebrate assemblages to direct measurements of near‐bed hydraulic conditions that integrate the complex three‐dimensional structure of flow close to the bottom. 2. We sampled invertebrate taxa from a Mediterranean River along a spatial gradient of increasing shear stress in two seasons (spring and autumn) with different hydrological conditions. We used a recently described ordination technique, Outlying Mean Index (OMI) analysis, to study the response of stream invertebrates to near‐bed hydraulic parameters. 3. The distribution of nearly 70% of the taxa collected was significantly related to the hydraulic parameters assessed. In both seasons, shear stress and Froude number were the most important hydraulic parameters whereas substratum particle size and bed roughness had less influence. Most of the 31 taxa collected in both seasons had a higher OMI (an index showing the deviation between the mean environmental conditions used by a taxon and the mean environmental conditions used by a theoretical taxon uniformly distributed across the studied gradient) in autumn (when flow was greater) and were found in samples with high shear stress and high Froude number. This suggests that benthic invertebrates changed their preferences according to flow conditions. 4. Taxon richness declined with increased shear stress during lower flow in spring. Finally, and agreeing with previous results, the proportion of filter feeders and collector‐gatherers was inversely related to shear stress. 5. Our results are a first step towards better habitat suitability models that could inform management decisions.     

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.     

The role of mechanical shear and cavitation on the behaviour of molds

The morphology of filamentous microorganisms does essentially affect the production of metabolites. Agitating conditions may affect the morphology and for this reason the production of metabolites too. The following parameters it was found to have an influence:

• Reynolds mixing number
• impeller blade tips velocity
• mean shear stress close to the impeller
• impeller power consumption per unit volume
• cavitation pressure drop

It were presumed three mechanisms for the mechanical effect on the microorganisms:

• 1 the direct impact of the impeller blades on the microorganisms-collision
• 2 the shear stress in the liquid phase
• 3 a sharp pressure decrease behind the impeller blades-cavitation

Mathematical relationships are developed for the different mechanisms.Using Aspergillus niger it is shown what morphological and physiological states of this microorganism are caused by mechanical straining and the conditions for the maximal production of citric acid are studied. Requirements for scale-up are discussed.     

Inter-individual variations in wall shear stress and mechanical stress distributions at the carotid artery bifurcation of healthy humans

Fluid shear stress and mechanical wall stress may play a role in the formation of early atherosclerotic lesions, but these quantities are difficult to measure in vivo. Our objective was to quantify these parameters in normal subjects in a clinical setting, and to define regions of low wall shear stress and high mechanical stress. The right carotid bifurcations of five healthy male volunteers were investigated using a novel non-invasive technique which integrates magnetic resonance angiography, ultrasonography, tonometry and state-of-the-art computational fluid dynamics and solid mechanics models. Significant inter-subject variations in patterns as well as magnitude of wall shear stress and mechanical stress were found. In spite of individual variabilities, this study revealed that some regions of the artery wall are exposed simultaneously to low wall shear stress and high mechanical stress and that these regions correspond to areas where atherosclerotic plaque develops. The coexistence of regions of low wall shear stress and high tensile stress may be an important determinant of the formation of atheroma in human arteries.