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Settling velocity is a crucial parameter in granular sludge technology. In this study the effects of temperature and salt concentrations on settling velocities of granular sludge particles were evaluated. A two-fold slower settling velocity for the same granules was observed when the temperature of water decreases from 40 °C to 5 °C. Settling velocities also decreased with increasing salt concentrations. Experiments showed that when granules were not pre-incubated in a solution with increased salt concentration, they initially floated. The time dependent increase in mass and hence in settling speed of a granule due to salt diffusion into the granule was dependent on the granule diameter. The time needed for full salt equilibrium with the bulk liquid took 1 min for small particles from the top of the sludge bed and up to 30 min for big granules from the bottom of the sludge bed. These results suggest that temperature and salt concentration are important parameters to consider in the design, start-up and operation of granular sludge reactors and monitoring of these parameters will aid in a better control of the sludge management in anaerobic and aerobic granular sludge technology. The observations also give an explanation for previous reports which were suggesting that a start-up of granular sludge reactors is more difficult at low temperatures.

The desired product of bioprocesses is often produced in particulate form, either as an inclusion body (IB) or as a crystal. Particle harvesting is then a crucial and attractive form of product recovery. Because the liquid phase often contains other bioparticles, such as cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is a complex process. In most cases, the particulate product is purified using selective solubilization or extraction. However, if selective particle recovery is possible, the already high purity of the particles makes this downstream process more favorable. This work gives an overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving forces that may be used for particle-particle separation, such as the centrifugal force, the magnetic force, the electric force, and forces related to interfaces. By coupling these driving forces to the resisting forces, the limitations of using these driving forces with respect to particle size are calculated. It shows that centrifugation is not a general solution for particle-particle separation in biotechnology because the particle sizes of product and contaminating particles are often very small, thus, causing their settling velocities to be too low for efficient separation by centrifugation. Examples of such separation problems are the recovery of IBs or virus-like particles (VLPs) from (microbial) cell debris. In these cases, separation processes that use electrical forces or fluid-fluid interfaces show to have a large potential for particle-particle separation. These methods are not yet commonly applied for large-scale particle-particle separation in biotechnology and more research is required on the separation techniques and on particle characterization to facilitate successful application of these methods in industry., ((c) 2006 Wiley Periodicals, Inc.)

Selective dissolved-air flotation for the separation of medium-chain-length polyhydroxyalkanoate (PHA) inclusion bodies (IBs) from Pseudomonas putida cell debris is investigated. Measurements show that both P. putida cell debris and PHA IBs have an iso-electric point of approximately pH 3.5. Selective aggregation and as a result selective flotation of PHA IBs was observed near this pH. Qualitative prediction of the aggregation behaviour was possible on the basis of the Van der Waals, hydrophobic and electrostatic interactions. In some cases however, the stability of the suspension could not be explained with these forces alone. It was therefore suggested that additional interactions, such as steric/brush effects, play an important role in the aggregation process.

The interfacial partitioning behavior of ampicillin and phenylglycine crystals in different two-phase systems has been investigated. The two-phase systems employed are water/dodecane, water/1-butanol, and water/pentane/methanol. By means of partition experiments and microscopic imaging, it has been shown that the mechanism of separation strongly depends on the choice of the two-phase system. While water/dodecane features a mechanism of sheer competitive adsorption at the interface, separation in water/1-butanol is mainly due to partitioning into both liquid phases, leading to a higher degree of separation. Experiments with water/pentane/methanol have illustrated the large potential of three-component systems, as slight variations in the composition can have large effects on the separation.

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.

Quantification of solid cell material (cell debris) is necessary for the optimisation of the efficiency of bioseparations. Cell debris can be quantified by detection of a component present in the cell wall that can act as a marker for cell debris. Membrane-associated proteins have previously been used as a marker for cell debris. This marker was quantified by SDS-PAGE with densiometry. In this paper cell debris quantification methods are presented that are faster and more accurate, i.e. membrane-associated protein quantification with the Protein 50 Labchip of Agilent Technologies, or that make use of peptidoglycan as marker for cell debris, i.e. a spectrophotometric muramic acid assay.

Foam disruption by agitation-the stirring as foam disruption (SAFD) technique-was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD-foam entrainment-was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised.

In this article, a qualitative study of the recovery of small bioparticles by interfacial partitioning in liquid-liquid biphasic systems is presented. A range of crystallised biomolecules with varying polarities have been chosen such as glycine, phenylglycine and ampicillin. Liquid-liquid biphasic systems in a range of polarity differences were selected such as an aqueous two-phase system (ATPS), water-butanol and water-hexanol. The results indicate that interfacial partitioning of crystals occurs even when their density exceeds that of the individual liquid phases. Yet, not all crystals partition to the same extent to the interface to form a stable and thick interphase layer. This indicates some degree of selectivity. From the analysis of these results in relation to the physicochemical properties of the crystals and the liquid phases, a hypothetical mechanism for the interfacial partitioning is deduced. Overall these results support the potential of interfacial partitioning as a large scale separation technology., (Copyright 2002 Wiley Periodicals, Inc.)

The hydrodynamics and mass transfer, specifically the effects of gas velocity and the presence and type of solids on the gas hold-up and volumetric mass transfer coefficient, were studied on a lab-scale airlift reactor with internal draft tube. Basalt particles and biofilm-coated particles were used as solid phase. Three distinct flow regimes were observed with increasing gas flow rate. The influence of the solid phase on the hydrodynamics was a peculiar characteristic of the regimes. The volumetric mass transfer coefficient was found to decrease with increasing solid loading and particle size. This could be predominantly related to the influence that the solid has on gas hold-up. The ratio between gas hold-up and volumetric mass transfer coefficient was found to be independent of solid loading, size, or density, and it was proven that the presence of solids in airlift reactors lowers the number of gas bubbles without changing their size. To evaluate scale effects, experimental results were compared with theoretical and empirical models proposed for similar systems., (Copyright 1998 John Wiley & Sons, Inc.)

Generalizing results from fungal fermentations is difficult due to their high sensitivity toward slight variation in starting conditions, poor reproducibility, and difference in strains. In this study a mathematical model is presented in which oxygen transfer, agitation intensity, dissolved oxygen tension, pellet size, formation of mycelia, the fraction of mycelia in the total biomass, carbohydrate source consumption, and biomass growth are taken into account. Two parameters were estimated from simulation, whereas all others are based on measurements or were taken from literature. Experimental data are obtained from the fermentations in both 2 L and 100 L fermentors at various conditions. Comparison of the simulation with experiments shows that the model can fairly well describe the time course of fungal growth (such as biomass and carbohydrate source concentrations) and fungal morphology (such as pellet size and the fraction of pellets in the total biomass). The model predicts that a stronger agitation intensity leads to a smaller pellet size and a lower fraction of pellets in the total biomass. At the same agitation intensity, pellet size is hardly affected by the dissolved oxygen tension, whereas the fraction of mycelia decreases slightly with an increase of the dissolved oxygen tension in the bulk. All of these are in line with observations at the corresponding conditions., (Copyright 1998 John Wiley & Sons, Inc.)

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%., (Copyright 1998 John Wiley & Sons, Inc.)

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.

Quantification of yeast flocculation under defined conditions will help to understand the physical mechanisms of the flocculation process used in beer fermentation. Flocculation was quantified by measuring the size of yeast flocs and the number of single cells. For this purpose, a method to measure floc size and number of single cells in situ was developed. In this way, it was possible to quantify the actual flocculation during fermentation, without influencing flocculation. The effects of three physical parameters, floc strength, fluid shear, and yeast cell concentration, on flocculation during beer fermentation, were examined. Increasing floc strength results in larger flocs and lower numbers of single cells. If the fluid shear is increased, the size of the flocs decreases, and the number of single cells remains constant at approximately 10% of the total cells present. The cell concentration also influences flocculation, a reduction of 50% in cell concentration leads to a decrease of about 25% in floc size. The number of single cells decreases in linear proportion to the cell concentration. This means that, during yeast settling at full scale, the number of single cells decreases. The results of this study are used in a model for yeast flocculation. With respect to full scale fermentation the effect of cell concentration will play an important role, for flocculation and sedimentation will occur simultaneously leading to a quasi steady state between these phenomena. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 190-200, 1997.

Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 +/- 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 +/- 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.

Mass transfers of O(2), CO(2), and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, k(L)a measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H(2)SO(4) in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70 degrees C, (k(L)a)(o) values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (k(L)a)(o) was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (k(L)a)(o) was plotted against the temperature. By multiplying the equilibrium concentration of O(2) in water with (k(L)a)(o) maximal, O(2) transfer capacities were calculated. It appeared that in finite of a decreased solubility of O(2) at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70 degrees C. However, in an air-mixed fermentor the transfer capacity of O(2) decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO(2) transfer capacity, calculated as the product of the equilibrium CO(2) concentration times (k(L)a)(c), decreased slowly as the temperature increased over the range 15-70 degrees C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO(2) limitation is more likely to occur than O(2) limitation.