New Insights into the Pelletization Mechanism by Extrusion/Spheronization

Pellet manufacturing by extrusion/spheronization is quite common in the pharmaceutical field because the obtained product is characterized by a high sphericity as well as a narrow particle size distribution. The established mechanisms only consider deformation of the initially fractured particles but do not account for mass transfer between the particles as a factor in achieving spherical particles. This study dealt with the visualization of mass transfer during spheronization. Therefore, two common pelletization aids, microcrystalline cellulose and kappa-carrageenan, were used alone as well as in combination with lactose as a filler. This study proves that mass transfer between particles must be considered in addition to plastic deformation in order to capture the spheronization mechanism. Moreover, it is evident that there are regional distinctions in the amount of mass transfer at the particle surface. Therefore, the commonly espoused pelletization mechanisms need to be extended to account for material transfer between pellet particles, which has not been considered before.     

Diffusion and reaction within porous packing media: a phenomenological model

A phenomenological model has been developed to describe biomass distribution and substrate depletion in porous diatomaceous earth (DE) pellets colonized by Pseudomonas aeruginosa. The essential features of the model are diffusion, attachment and detachment to/from pore walls of the biomass, diffusion of substrate within the pellet, and external mass transfer of both substrate and biomass in the bulk fluid of a packed bed containing the pellets. A bench-scale reactor filled with DE pellets was inoculated with P. aeruginosa and operated in plug flow without recycle using a feed containing glucose as the limiting nutrient. Steady-state effluent glucose concentrations were measured at various residence times, and biomass distribution within the pellet was measured at the lowest residence time. In the model, microorganism/substrate kinetics and mass transfer characteristics were predicted from the literature. Only the attachment and detachment parameters were treated as unknowns, and were determined by fitting biomass distribution data within the pellets to the mathematical model. The rate-limiting step in substrate conversion was determined to be internal mass transfer resistance; external mass transfer resistance and microbial kinetic limitations were found to be nearly negligible. Only the outer 5% of the pellets contributed to substrate conversion. (c) 1993 Wiley & Sons, Inc.     

Mathematical analysis of solid-liquid mass transfer in a batch reactor for the enzymatic transformation of testosterone to 4AD

A previous mathematical analysis of mass transfer in a two-phase (solid-liquid) batch reactor for enzymatic transformation of testosterone to 4AD (Pereira et al., 1987) is extended to incorporate the effect of convective mixing. The results of the analysis showed that for a given enzyme loading, the mass transfer resistance in the solid (a function of the bead size) and the intensity of convective mixing (as embodied in the mass transfer coefficient) are two parameters that can be varied such that the overall mass transfer rate from the solid to the liquid phase ensures optimal reactor performance.     

Hydrodynamic and kinetic study of cellulase production by Trichoderma reesei with pellet morphology

Numerical simulations and experimental validation were performed to understand the effects of hydrodynamics on pellet formation and cellulase production by filamentous T. reesei. The constructed model combined a steady-state multiple reference frame (MRF) approach describing mechanical mixing, oxygen mass transfer, and non-Newtonian flow field with a transient sliding mesh approach and kinetics of oxygen consumption, pellet formation, and enzyme production. The model was experimentally validated at various agitation speeds in a two-impeller Rushton turbine fermentor. Results from simulation and experimentation showed that higher agitation speeds led to increases in the pellet diameter and the proportion of pelletized (vs. filamentous) forms of the biomass. It also led to increase in dissolved oxygen mass transfer rate in shear-thinning fluid and cellulase productivity. The extent of these increases varied considerably among agitation speeds. Pellet formation and morphology were presumably affected within a viscosity-dependent shear-rate range. Cellulase activity and cell viability were shown to be sensitive to impeller shear. A maximum cellulase activity of 3.5 IU/mL was obtained at 400 rpm, representing a twofold increase over that at 100 rpm.     

Analysis of the transient response of a CSTR containing immobilized enzyme particles: Part I. Model development and analysis of the influence of operating conditions and process parameters

The transient response of the bulk substrate concentration in a CSTR containing immobilized enzyme (IMEs) in porous solid supports and the possibilities of exploiting the minimum behavior for parameter estimation purposes are studied in this work. For that purpose, mathematical models have been developed for several kinetic expressions with parallel deactivation mechanisms and for two different particle shapes (cylindrical and spherical). The influence of a number of system variables and non-dimensional parameters, i.e., pellet radius, mass of particles, flow rate, effective diffusivity, mass transfer coefficient, Thiele modulus, Michaelis constant, and enzyme deactivation constant, is also reported in a systematic way. Simulations, using realistic data, show that the minimum bulk substrate concentration is sufficiently pronounced and the time scale for the minimum to occur is sufficiently practical, so that it can be used to extract several parameters of the immobilized enzyme system.     

A method to evaluate the isothermal effectiveness factor for dynamic oxygen into mycelial pellets in submerged cultures

Several models have been developed simulating O2 transfer in bioreactors, but three limitations are often found: (i) an inadequate kinetic representation of O2 consumption or wrong boundary conditions, (ii) unrealistic parameter values, and (iii) inadequate experimental systems. In our study we minimized those possible sources of error. Oxygen uptake rate, void fraction of the pellet, and external O2 mass transfer coefficient were experimentally obtained from bioreactor studies in which pellets of Gibberella fujikuroi were naturally formed. Michaelis-Menten kinetics and diffusion equations were used to describe the O2 consumption rate and to evaluate the effectiveness factor in dynamic mode. The nonlinear mathematical model proposed was solved by the orthogonal collocation technique. The O2 consumption rate in pellets of G. fujikuroi of 1.7-2.0 mm is only marginally inhibited by diffusion constraints under conditions tested. Simulation analysis showed that the effectiveness factor decreased as the Thiele modulus and pellet diameter increased. The proposed model was applied to experimental data reported for other fungal pellets and allowed to predict optimal conditions for O2 transfer into mycelial pellets.     

Strategies for penicillin fermentation in tower-loop reactors

Since it has not been possible to produce penicillin in tower-loop reactors with highly viscous filamentous molds of Penicillium chrysogenum which are employed in stirred-tank reactors, a new strategy has been developed to avoid the formation of this morphology and to use the pellet form of the fungi. When employing definite impeller speeds in the subculture in connection with definite inoculum amounts and substrate concentrations in the main culture (bubble column), it is possible to generate a suspension of isolated small pellets, which shows a low broth viscosity up to a sediment content of 45% over the entire fermentation time. Volumetric mass-transfer coefficients k(L)as are by a factor of 4 to 5 higher in these pellet suspensions than in filamentous broths. It was easy to maintain the necessary oxygen supply for penicillin production in these pellet suspensions. Under these conditions the specific penicillin productivities were higher with regard to power input (up to 90%), biomass, and consumed substrate than in the stirred-tank reactors with highly viscous filamentous morphology of the fungi. Under nonoptimized operating conditions the absolute penicillin production in the tower loop was 35% lower than in the stirred-tank reactor due to lower possible biomass concentrations. The separation of the biomass, and therefore the penicillin recovery, is much simpler when employing pellets. It is shown how the particular mass transfer resistances at the gas/liquid and liquid/pellet interfaces and within the pellets change with the pellet diameter. There should be a particular pellet diameter at which penicillin productivity has its maximum. These investigations indicate that the use of tower-loop reactors can, in the future, be an alternative for more economical penicillin production methods.     

Verbesserung alter biotechnologischer Verfahren

By the example of the Penicillin G production, it is shown, how the production process can be improved by using pellet suspension instead of highly viscous filamentous mycel. In order to optimize the pellet geometry, dissolved oxygen concentration profiles were measured, and the overall mass transfer resistance was calculated in the pellets; histological investigations were carried out with pellets also. All measurements indicated that the optimal pellet diameter is 400 μm. When using this pellet, the specific power input was reduced from 4–5 kW/m³ to 0.8 kW/m³. The losses during the recovery of Penicillin G can be reduced below 1% by using reactive extraction. The thermodynamics, kinetics and reaction technique of reactive extraction of Penicillin G are also taken into account.     

Quantification of Mass Transfer During Spheronisation

Spherical granules (pellets) are quite useful in many pharmaceutical applications. The extrusion spheronisation technique is well established as a method of producing pellets of a spherical shape and narrow size distribution. After the extrusion, the cylindrical extrudates are transformed to spherical pellets by spheronisation. The frequently used models consider deformation and breakage during this process. However, the adhesion of fine particles has been neglected as a mechanism in spheronisation for many years. This study quantifies the mass transfer between pellets during spheronisation. During the investigation, the pelletisation aids (microcrystalline cellulose and kappa-carrageenan), the drug (acetaminophen and ibuprofen) and water content were varied systematically. A novel parameter, namely, the "mass transfer fraction" (MTF), was defined to quantify the mass transfer between the pellets. All four investigated formulations had an MTF between 0.10 and 0.52 that implies that up to 50 % of the final pellet weight was involved in mass transfer. Both pelletisation aids showed similar MTF, independent of the drug used. Furthermore, an increase of the MTF, with respect to an increase of the water content, was found for microcrystalline cellulose formulations. In conclusion, the mass transfer between the pellets has to be considered as a mechanism for spheronisation.KEY WORDS: carrageenan, MCC, mechanism, pellets, spheronisation     

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (_a) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml^–1 and 0.9–2.1 g l^–1 dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm³ was observed in cultures supplemented with 3 g l^–1 xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the _a. Counter-intuitively, an increase in the _a within the experimental range examined resulted in an increase in the rate of gas–liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.     

Effect of pellet size and pellet quality on pig performance

An experiment involving 1360 growing finishing pigs was undertaken to examine the effect of pellet size and pellet quality, as measured by the Holmen pellet durability test, on pig performance. A barley and soya bean meal diet was used and pellets were of two sizes, 5 and 10 mm diameter. Pellet quality was varied to give two types of pellet by steam conditioning and screening procedure during the pelleting process. The mean difference obtained in pellet durability was 11%.

Pig performance between 30 and 80 kg liveweight was not affected by either pellet size or pellet quality. There were small non-significant trends in favour of both the smaller pellets and the lower quality pellets. These trends, of the order of 1% or less, followed the same pattern as the dry matter content of the diets. The smaller diameter pellets were dried more efficiently in the cooling process and the low durability pellets had less steam added during the manufacturing process, which was reflected in the dry matter content of the finished diets.     

Morphological characterization of filamentous microorganisms in submerged cultures by on-line digital image analysis and pattern recognition

The morphology of filamentous microorganisms in submerged culture is of great interest. On the one hand, morphology influences rheology and mass transfer in the fermentation broth. On the other hand, morphology could be a visible expression of physiology and metabolism of the microorganisms. An algorithm for the morphological characterization and the estimation of biomass of filamentous microorganisms by means of digital image analysis has been developed. After measurement of eight features the objects in the broth are classified into different morphological classes, i.e., pellet aggregates, rough pellets, smooth pellets, mycelial flocks, and medium components. The classification is based on the measured object parameters and a knowledge base, which was generated in a preceding training phase. The method was tested on Streptomyces tendae Tü 901/8c. A typical batch fermentation in a defined medium is presented. It could be shown that both morphology and physiology have been changed in the course of the fermentation, especially during the transition from trophophase to idiophase. In order to supervise the fermentation processes continuously, an on-line image analysis system has been developed. Sampling, dilution, and image acquisition of the culture were performed under the control of a personal computer. (c) 1997 John Wiley & Sons, Inc.     

Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase

The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.     

Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest?, and their combinations for shotgun mass spectrometry. An initial proteomic analysis was performed following urea, GnHCl, and RapiGest? extraction of equine superficial digital flexor tendon (SDFT) tissue. Subsequently, another proteomic analysis was performed following extraction with GnHCl, Rapigest?, and their combinations. Between the two chaotropic agents, GnHCl extracted more proteins, whilst a greater number of proteins were solely identified after Rapigest? extraction. Protein extraction with a combination of GnHCl followed by RapiGest? on the insoluble pellet demonstrated, after label‐free quantification, increased abundance of identified collagen proteins and low sample to sample variability. In contrast, GnHCl extraction on its own showed increased abundance of identified proteoglycans and cellular proteins. Therefore, the selection of protein extraction method for tendon tissue for mass spectrometry analysis should reflect the focus of the study.     

A novel rectangular airlift reactor with mesh baffle-plates

An experimental rectangular airlift reactor having mesh baffle-plates has been fabricated out of Perspex and compared with conventional airlift and bubble column in terms of gas holdup, volumetric mass transfer coefficient and mixing time. Mesh baffle-plates improved mass transfer and mixing with the mass transfer coefficient of the proposed reactor being up to 12% higher than that in a conventional airlift reactor under the same operating condition. The mixing time of the proposed reactor can be 95% lower than that of the airlift reactor.     

Continuous production of citric acid with recirculation of the fermentation broth after product recovery

An airlift reactor with double net draft tubes was developed. A sparger was located between the two draft tubes. The draft tubes had a significant effect on breaking bubbles into smaller ones. The assessment of the reactor performance was based on gas holdup, mixing time, and volumetric mass transfer coefficient. The proposed reactor had higher gas holdup and volumetric mass transfer coefficient, and lower mixing time in comparison with those of the bubble column. Application of the proposed reactor to fermentation of Saccharomyces cerevisiae demonstrated that the cultivation time was significantly shortened.     

Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet

The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.     

Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation

The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation.     

Effect of stirrer speed,aeration rate and cell mass concentration on volumetric oxygen transfer coefficients (KLa) in the cultivation of Curvularia lunata in a batch reactor

Summary Curvularia lunata was grown in a stirred and aerated reactor for the production of extracellular rifamycin oxidase. Volumetric oxygen transfer coefficients (K_La) were measured for various stirrer speeds, rates of aeration and cell mass concentrations in the reactor. Stirrer speed and aeration rate were optimized and it was found that stirrer speeds of 400–500 rpm and aeration rates of 0.75–1 vvm were optimum for the maximum amount of enzyme production. It was noticed that the increase in cell mass decreased the oxygen transfer coefficient. It was also noticed that the organism formed pellets rather than mycelia when grown on glucose and with an increase in the concentration of glucose in the reactor, there was heavy pellet formation.