Morphological measurements on Penicillium chrysogenum broths by rheology and filtration methods

The morphology parameters of mycelial culture (Penicillium chrysogenum) were measured and quantified by rheology and filtration methods. Two of the morphology parameters obtained from rheology measurements, delta defined by the Casson equation and delta* defined by intrinsic viscosity, were found to vary systematically with broth age and with the observed morphology by microscopy. Three of the filtration parameters, hyphal density, Kozeny constant, and index of compressibility, are demonstrated as sensitive indicators of the broth age and mycelial morphology. Two of the morphology parameters, delta and delta*, were used to cross-correlate with hyphal density. Because various mycelial fermentations require different growth morphologies (pellet and filament) for optimum product yield and the morphology of mycelial broths varies with broth age, it is suggested that these morphology parameters could be used to represent the morphology of mycelial broths quantitatively. (c) 1993 John Wiley & Sons, Inc.     

A new sensor,the “filtration probe,” for quantitative characterization of the penicillin fermentation. II. The monitor of mycelial growth

Biomass concentration during penicillin production can be estimated rapidly and with reasonable accuracy using the “filtration probe.” The basis for the measurement is the volume of the filter cake which, in contrast to the conventional “packed-cell volume” measured by centrifugation, is only moderately affected by the morphology of the mycelia. The influence of the mycelial morphology on the specific filter cake volume [ υ (cm³ cake/g dry cells)] can additionally, to a large extent, be predicted from the (specific) filtration resistance of the broth. During the rapid (exponential) growth period, the specific cake volume is, however, more variable and typically exhibits a transient maximum value. The presence of such a maximum can be explained by a simple model for exponential growth of single mycelial particles. this model is based upon conventional kinetics for hyphae elongation and branching and applies well to experimental data (filtration behavior as well as microscopic studies) provided hyphae fragmentation is negligible (i.e., at high growth rates, μ ? μ_max).     

Effects of Carbon dioxide on the Rheological behavior and oxygen transfer in submerged penicillin fermentations

Fermentations of Penicillium chrysogenum have been made with different CO(2) contents in the influent gas streams. The rheological behavior of the culture broth was found to be significantly changed by exposure to high levels of CO(2). This is attributed to the wide variation in the morphology of P. chrysogenum, from normal mycelia with long hyphae to roughly spherical pellets when subjected to high levels of CO(2). A correlation has been developed relating volumetric O(2) transfer coefficients, k(L)a, with the effective O(2) diffusion coefficients, D(e), and the apparent viscosities, mu(app), based on the results obtained in this study. The use of CO(2) as a potent means for altering the rheological properties of culture broths and consequently improving the O(2) transfer capabilities in penicillin fermentations was clearly demonstrated.     

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.     

Oxygen transfer in slurry bioreactors

The oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40-L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re-examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.     

A fractal model for the characterization of mycelial morphology

A new technique based on a fractal model has been developed for the quantification of the macroscopic morophology of mycelia. The morphological structuring is treated as a fractal object, and the fractal dimension, determined by an ultrasonic scattering procedure developed for the purpose, serves as a quantitative morphological index. Experimental observations reported earlier and simulations of mycelial growth, carried out using a probabilistic-geometric growth model developed for the purpose, both validate the applicability of the fractal model. In experiments with three different species, the fractal dimensions of pelletous structures were found to be in the range 1.45-2.0 and those of filamentous structures were in the range 1.9-2.7, with values around 2.0 representing mixed morphologies. Fractal dimensions calculated from simulated mycelia are in rough agreement with these ranges. The fractal dimension is also found to be relatively insensitive to the biomass concentration, as seen by dilution of the original broths. The relation between morphology and filtration properties of the broths has also been studied. The fractal dimension shows a strong correlation with the index of cake compressibility and with the Kozeny constant, two filtration parameters that are known to be morphology dependent. This technique could thus be used to develop correlations between the morphology, represented by the fractal dimension, and important morphology-dependent process variables. (c) 1993 John Wiley & Sons, Inc.     

Estimation of the rheology of glucoamylase fermentation broth from the biomass concentration and shear conditions

Fermentation broths from nine Aspergillus awamori cultivations, carried out under different agitation and aeration conditions and distinct initial substrate concentrations, were characterized using a continuous on-line rheometer. A single correlation between the consistency index (K) and the biomass concentration (X), including the agitation (N) and aeration (Q) conditions fitted the experimental data reasonably well, showing that the shear conditions imposed on the broth influence in a significant way the mycelial morphology and, therefore, the broth rheology.     

Rheology of filamentous fermentations

The performance of a bioreactor containing a filamentous fermentation broth is greatly influenced by the rheological properties of the broth. These properties are determined mainly by the concentration of biomass, its growth rate and morphology. Included in the morphology are such factors as the geometry of hyphae (length, diameter, branching frequency), hyphal flexibility and hyphal-hyphal interactions, which can all be affected by the operational design of the reactor. Thus, correlations describing viscosity as a function of biomass only are of limited value. A better understanding of the relations between morphology and rheology may be achieved by a combination of rheological and morphological studies.Rheological properties are normally determined using off-line measurements in-spite of associated problems with sample treatment influencing the results. Equipment for dynamic, on-line, measurement of morphology and rheology is available, but little used in filamentous fermentations. Controlling the rheological properties of mycelial fermentations may be difficult because of the great number of factors influencing mycelial development and/or hyphal-hyphal interactions.Polymer solutions are often used to simulate flow behaviour of filamentous fermentations and scale-up and mass transfer considerations are based on these studies. Although much information has been gained this way, the predictions developed do not include the effect of an active biomass on the mass transfer and flow properties of the culture. It is important to carry out studies on the non-homogeneous fermentation fluids, and develop correlations based on these studies.     

Oxygen transfer in broths of plant cells at high density

The rheological properties of the culture broths of some plant cells (Cudrania tricuspidata, Vinca rosea, and Agrostemma githago) at high density (10-18 g dry wt/L) were measured, and oxygen transfer in the broths in various bioreactors was investigated. The rheological properties of the broths were dependent on the size, specific gravity, and concentration of the cell aggregates contained in the broths. The broths were non-Newtonian and pseudoplastic fluids. The flow behavior index n was fairly constant (0.53) and the consistency index K varied in proportion to the sixth-to-seventh power of the cell mass concentration M. The apparent viscosity mu(a) of the broths was in proportion to the 6.5th power of M. The oxygen transfer in the broths was discussed on the basis of the results obtained for suspensions of granulated agars (agar concentration, 5.8%) in water, which were similar to the broths in rheological properties. The volumetric oxygen transfer coefficient k(L)a in the broths was dependent on mu(a)(k(L)a proportional, variant mu(a) (-m)) and decreased greatly at a certain apparent viscosity, mu(ac). The values of m and mu(ac) were closely related to the aeration-agitation mechanisms of the bioreactors. The values of mu(ac) in aeration-agitation type bioreactors was larger than that in aeration-type bioreactors, whereas for m, the reverse was true.     

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.     

Influence of n-hexadecane on penicillin fermentation with Penicillium chrysogenum: The morphological effect

Summary In addition to acting as an oxygen transfer enhancer, n-hexadecane plays an important role in dominating the mycelial morphology of Penicillium chrysogenum in a submerged penicillin fermentation. The cell morphology is related to the timing of adding n-hexadecane to the culture. n-Hexadecane also has a function of suppressing foaming and thus can be employed as an antifoam agent in the fermentation.     

Oxygen transfer to mycelial fermentation broths in an airlift fermentor

Oxygen transfer rates and gas holdups were measured in mycelial fermentation broths of Chaetomium cellulolyticum and Neurospora sitophila, each cultured in a 1300-L pilot-plant-scale airlift fermentor. These cultures exhibited highly non-Newtonian flow behavior coupled with a substantial decrease in oxygen transfer rates. The volumetric mass transfer coefficients in these cultures were found to be 65-70% lower than those in water. The data were compared with the available correlations obtained for simulated fermentation broths. In general, the data for C. cellulolyticum are in satisfactory agreement with the correlations for the model media but the data for N. sitophila are higher than that predicted by the correlations. Model media based correlations are found to be applicable to the fermentation processes if the culture medium does not possess a high yield stress.     

Studies on Cardiotonic Substances Produced by Microorganisms. (I)

The active substances such as cardiotonics or toxics which were produced in the cultured broths or contained in the mycellia of microorganisms were examined by Ito’s assay method for cardiotonica using the embryonic heart of Japanese killi-fish.

There were some strains obtained from actinomycetes whose cultured broths or methanol mycelial extracts showed these activities, but fewer from moulds, yeasts and bacteria.     

Effect of carbon source and aeration rate on broth rheology and fungal morphology during red pigment production by Paecilomyces sinclairii in a batch bioreactor

The influence of carbon source and aeration rate on fermentation broth rheology, mycelial morphology and red pigment production of Paecilomyces sinclairii was investigated in a 5-l stirred-tank bioreactor. The characteristics of P. sinclairii grown on starch and on sucrose medium were comparatively studied: the specific growth rate in sucrose medium (0.04 h(-1)) was higher than that in starch medium, whereas the specific production rate of red pigments (0.04 gg(-1)d(-1)) was favorable in starch medium. P. sinclairii grown in sucrose medium were highly branched and showed longer hyphal lengths than that in starch medium. The consistency index (K) in sucrose medium was markedly higher than that in starch medium due to higher cell mass, while the higher values of flow behavior index (n) were indicated at the late stationary phase in starch medium. The aeration rate was varied within the ranges from 0.5 to 3.5 vvm while running the fermentation at mild agitation of 150 rpm using sucrose as the carbon source. The maximum biomass concentration of P. sinclairii was about 33 gl(-1) with an aeration rate of 1.5 vvm, whereas the maximum yield of red pigment production (4.73 gl(-1)) was achieved with 3.5 vvm. The highly branched cell morphology appeared at 1.5 vvm and the highly vacuolated cell morphology was observed in a high aeration rate (3.5 vvm). There was no significant variance in rheological parameters (K and n) between culture broths from different aeration conditions.     

Correlation of Aspergillus niger broth rheological properties with biomass concentration and the shape of mycelial aggregates

Aspergillus niger was grown in a 7-L chemostat at biomass levels of 7 to 9 gL(-1); dilution rates of 0.03, 0.05, 0.075, and 0.009 h(-1); and dissolved oxygen tensions of 7%, 12%, and 40% of air saturation. Broth rheological measurements were made on-line, while off-line image analysis was used to measure mycelial morphology, including characterization of mycelial aggregates (clumps). Under all conditions, more than 87% of the hyphase were in clumps, the shape of which determined the rheological characteristics of the broth. In particular, the power law consistency index could be correlated with the biomass concentration and the roughness factor of the clumps, which describes their hairiness. A decrease in specific growth rate decreased roughness, possibly due to changes in the amount of clump breakup. However, decreases of roughness with increasing dissolved oxygen tension might rather imply some effect on hyphal-hyphal interactions within the clumps. (c) 1993 John Wiley & Sons, Inc.     

Bubble rise velocities and drag coefficients in non-Newtonian polysaccharide solutions.

Microbially produced polysaccharides have properties which are extremely useful in different applications. Polysaccharide producing fermentations start with liquid broths having Newtonian rheology and end as highly viscous non-Newtonian solutions. Since aerobic microorganisms are used to produce these polysaccharides, it is of great importance to know the mass transfer rate of oxygen from a rising air bubble to the liquid phase, where the microorganisms need the oxygen to grow. One of the most important parameters determining the oxygen transfer rate is the terminal rise velocity of air bubble. The dynamics of the rise of air bubbles in the aqueous solutions of different, mostly microbially produced polysaccharides was studied in this work. Solutions with a wide variety of polysaccharide concentrations and rheological properties were studied. The bubble sizes varied between 0.01 mm3 and 10 cm3. The terminal rise velocities as a function of air bubble volume were studied for 21 different polysaccharide solutions with different rheological properties. It was found that the terminal velocities reached a plateau at higher bubble volumes, and the value of the plateau was nearly constant, between 23 and 27 cm/s, for all solutions studied. The data were analyzed to produce the functional relationship between the drag coefficient and Reynolds number (drag curves). It was found out that all the experimental data obtained from 21 polysaccharide solutions (431 experimental points), can be represented by a new single drag curve. At low values of Reynolds numbers, below 1.0, this curve could be described by the modofoed Hadamard-Rybczynski model, while at Re > 60 the drag coefficient was a constant, equal to 0.95. The latter finding is similar to that observed for bubble rise in Newtonian liquids which was explained on the basis of the "solid bubble" approach.     

Cellulose degradation and monitoring of viscosity decrease in cultures of Cellulomonas uda grown on printed newspaper

The rheological behavior of cultures of Cellulomonas uda with shredded printed newspaper as the carbon source was studied. The initial substrate concentrations ranged from 23 to 60 g/L. The changes in apparent viscosity were followed on-line by applying a commercially available process viscometer and discretely using a rotational viscometer with an anchor impeller. During the time of highest cellulose degradation, the broths exhibited a pseudoplastic behavior which could be explained satisfactorily by the power-law model. At the end of cultivation when cellulose degradation slowed down, the broths became Newtonian in behavior. Endo-1,4-beta-glucanase, 1,4-beta-xylanase, beta-glucosidase, and beta-xylosidase activities were also determined during cultivation as well as cellulose degradation and cell mass production. The beginning of endoglucanase formation and the start of the final viscosity decrease of the bacterial paper pulp suspensions could be correlated.     

Broth rheology and morphological analysis of Solanum chrysotrichum cultivated in a stirred tank

Solanum chrysotrichum cell cultures were grown in a stirred tank bioreactor and their rheological and morphological behaviour were evaluated. The culture broths exhibited non-Newtonian and shear-thinning characteristics. Pseudoplasticity of the broths was governed by their biomass concentration. The roundness of aggregates measured as the elliptical form factor (EFF) had important changes. At the beginning of the culture the aggregates with an EFF lower than 2 represented 52% of the population, but in stationary phase the proportion increased to 77%. Whereas the size of aggregates did not change 80% of the population had an area lower than 0.1 mm². Overall, these results indicate that the shape of the aggregate therefore needs to be considered when studying plant broth rheology.     

High-performance liquid chromatographic identification of beta-lactam antibiotics produced by dermatophytes

Thirteen of 48 dermatophyte isolates were found by bioassay to produce beta-lactam antibiotics and seven produced other antibiotics. Estimation and detection of specific beta-lactams in culture broths by derivatization and HPLC was only possible following concentration and extraction procedures. Analysis of the concentrated broths demonstrated the production of penicillin X and penicillin G by two Trichophyton mentagrophytes strains and by one Microsporum canis strain; one further T. mentagrophytes strain produced only penicillin X. Additions of the beta-lactam side-chain precursors, phenylacetic acid and phenoxyacetic acid, to fermentation media failed to increase the antibiotic titres.