The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum

Penicillium chrysogenum strain P1 was grown on complex media in 10 and 100 L agitated fermenters at various aeration rates and stirrer speeds. Samples were removed at intervals for measurements of the culture morphology. At high stirrer speeds (1000 and 1200 rpm) in 10-L fermentations the rate of decrease in the mean effective hyphal length was faster and the rate of penicillin production was lower than fermentations done at 800 rpm. At similar power inputs per unit volume in 100-L fermentations, the change in mean effective hyphal length was less and higher penicillin production rates were observed. This work comparing the results at two scales has shown that neither of the concepts of impeller tip speed or the dissipation rate of turbulence have general validity as a measure of hyphal damage. Our results are reasonaby well correlated by groups similar to circulation rate (ND(i) (3)/V) with lower circulation rates being beneficial. An adaptation of the van Suijdam and Metz relationship, expressed as P/D(i) (3)t(c), was most successful. Our data are insufficient to demonstrate the generality of the relationship but do support the concept of a dispersion zone around the impellers in which mycelia may be damaged. The greater the frequency of circulation of mycelia through the zone the greater the damage and the lower the rate of penicillin synthesis by the culture.     

Bioreactor magnetic fields do not affect growth and antibody productivity of GAP-A3 hybridomas

Summary GAP-A3 hybridoma cells grown in T-flasks exposed to the rotating magnetic field of a spinner flask stirrer base show no effect of stirrer speed in the range 0–150 rpm on their growth rate or antibody productivity. Similarly mechanically and magnetically driven spinner flasks at 48 and 70 rpm show no difference in these rates that may be attributed to the magnetic field.     

Effect of shear on morphology and erythromycin production in Saccharopolyspora erythraea fermentations

A complex medium was used to investigate the effects of shear on the S. erythraea fermentation at 7-l scale. Maximum biomass was 11.1 - 0.5 g l^у at 1250 rpm (tip speed = 4.45 ms^у), whereas it was 12.7 - 0.2 g l^у at 350 rpm (tip speed = 1.07 ms^у). Specific erythromycin production was not stirrer speed dependent in the range of 350 to 1000 rpm and decreased by 10% at stirrer speed of 1250 rpm. Morphological measurements using image analysis showed that the major axis of the mycelia (both freely dispersed and clumps) decreased after the end of the rapid growth phase to a relatively constant value (equilibrium size) dependent on the stirrer speed. The mechanical properties of the cell wall were examined by disruption of fermentation broth in homogeniser and it was shown that mechanical strength of the cell wall increased in a large extent during deceleration phase.     

Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30

Trichoderma reesei was grown in a stirred-tank bioreactor (STB) and a reciprocating plate bioreactor (RPB) at four different agitation speeds. A semiautomatic image analysis protocol that was developed to characterize the mycelium morphology during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms) was applied to study the effect of agitation on the morphology of T. reesei. It was shown via statistical validation that broth samples used for image analysis represented the whole population of the fungi in the bioreactor. High shear was found to be damaging to T. reesei grown in the STB. The gentler shear produced in the RPB was not detrimental to the microorganism even at higher agitation speed. Better productivity was obtained for T. reesei grown in the STB and the highest productivity, 0.121 IU/mL h, was obtained at 400 rpm. The morphological parameter, the hyphal growth unit, was found to be correlated to the productivity. Understanding the effect of agitation on the morphology and productivity of T. reesei could lead to the design of better bioreactors and the selection of operating conditions of bioreactors to optimize the production of cellulase.     

Influence of oxygen transfer on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> effects on growth rate and biodesulfurization capacity

The growth rate and desulfurization capacity accumulated by the cells during the growth of Pseudomonas putida KTH2 under different oxygen transfer conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different agitation conditions. During the culture, several magnitudes associated to growth, such as the specific growth rate, the dissolved oxygen concentration and the carbon source consumption have been measured. Experimental results indicate that cultures are influenced by the fluid dynamic conditions into the bioreactor. An increase in the stirrer speed from 400 to 700 rpm has a positive influence on the cell growth rate. Nevertheless, the increase of agitation from 700 to 2000 rpm hardly has any influence on the growth rate. The effect of fluid dynamics on the cells development of the biodesulfurization (BDS) capacity of the cells during growth is different. The activities of the intracellular enzymes involved in the 4S pathway change with dissolved oxygen concentration. The enzyme activities have been evaluated in cells at several growth time and different hydrodynamic conditions. An increase of the agitation from 100 to 300 rpm has a positive influence on the development of the overall BDS capacity of the cells during growth. This capacity shows a decrease for higher stirrer speeds and the activity of the enzymes monooxygenases DszC and DszA decreases dramatically. The highest value of the activity of DszB enzyme was obtained with cells cultured at 100 rpm, while this activity decreases when the stirrer speed was increased higher than this value.     

Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.     

The influence of preculture on the process performance of penicillin V production in a 100-l air-lift tower loop reactor

Summary The influence of stirrer speed in the third preculture on the performance of penicillin V production by Penicillium chrysogenum in complex medium in a 100-l air-lift tower loop reactor was investigated. The process performance in the main culture was improved by increasing the stirrer speed from 500 to 750 rpm: the pellet size was reduced to half, the cell growth was influenced only slightly, but the production phase was extended considerably, and the final penicillin concentration was increased from 5.1 g 1^-1 to 10.4 g 1^-1.     

Stirred Tank Two-Liquid Phase Biocatalytic Reactor Studies: Kinetics, Evaluation and Modelling of Substrate Mass Transfer

Kinetic measurements of the benzyl acetate hydrolysis by pig liver esterase in a two-liquid phase stirred tank reactor were made at a variety of aqueous phase enzyme concentrations and stirrer speeds. All experiments were performed in an inverted liquid-liquid system at a high phase ratio. The results were explained in terms of the aqueous phase bulk reaction model developed from previous Lewis cell studies. An algorithm is presented for the indirect measurement of the substrate mass transfer coefficient and consequently a model was developed to predict reaction rates. While the model describes the kinetics effectively, and could therefore be used to predict reactor behaviour, no difference was observed between kinetic measurements made at a stirrer speed of 750 and 1000 rpm.     

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus

Clavulanic acid (CA), a potent β-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O₂ (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O₂, were performed. Significant variations in CA titer, CA production rate and O₂ uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.     

Effect of shear rate and culture pH on the production of xylanase by Thermomyces lanuginosus

Summary The pH-value and the stirrer speed during cultivation of the thermophilic fungus Thermomyces lanuginosus were found to have a pronounced influence on xylanase production using corn cobs as carbon source. The highest xylanase activity of 32500 nkat/ml was produced in labscale fermentation within 118 hours at a stirrer speed of 50 rpm and a controlled pH-value of 7.5.     

Effects of preculture variability on clavulanic acid fermentation.

The production profile of clavulanic acid by Streptomyces clavuligerus was shown to be strongly dependent on inoculum activity. Two sets of fermentations (A and B) were investigated at industrial pilot-plant scale using complex media. Type A fermentations were inoculated using late exponential growth phase mycelia. Type B fermentations were inoculated using mycelia harvested at stationary phase. Productivities throughout type A fermentations were consistently higher than type B, reaching a maximum at about 70 h and then decaying to the same final productivities at 140 h of type B runs. Several scheduling alternatives, based on combinations of the two inocula types and different fermentation lengths, were compared in terms of the overall process economics (fermentation and downstream). An increase of ca. 22% on the overall process profit is predicted using late exponential growth phase inocula and a fermentation duration of only 96 h. A new operating strategy was thus proposed for inoculum production based on the control of preculture activity using off-gas analysis. This method ensures higher productivity and better batch-to-batch reproducibility of clavulanic acid fermentations than traditional methods based on constant age inocula.     

Dependence of penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity

The influence of the agitation conditions on the growth, morphology, vacuolation, and productivity of Penicillium chrysogenum has been examined in 6 L fed-batch fermentations. A standard Rushton turbine, a four-bladed paddle, and a six-bladed pitched blade impeller were compared. Power inputs per unit volume of liquid, P/VL, ranged from 0.35 to 7.4 kW/m3. The same fermentation protocol was used in each fermentation, including holding the dissolved oxygen concentration above 40% air saturation by gas blending. The mean projected area (for all dispersed types, including clumps) and the clump roughness were used to characterize the morphology. Consideration of clumps was vital as these were the predominant morphological form. For a given impeller, the batch-phase specific growth rates and the overall biomass concentrations increased with agitation intensity. Higher fragmentation at higher speeds was assumed to have promoted growth through increased formation of new growing tips. The mean projected area increased during the rapid growth phase followed by a sharp decrease to a relatively constant value dependent on the agitation conditions. The higher the speed, the lower the projected area for a given impeller type. The proportion by volume of hyphal vacuoles and empty regions decreased with speed, possibly due to fragmentation in the vacuolated regions. The specific penicillin production rate was generally higher with lower impeller speed for a given impeller type. The highest value of penicillin production as well as its rate was obtained using the Rushton turbine impeller at the lowest speed. At given P/VL, changes in morphology, specific growth rate, and specific penicillin production rate depended on impeller geometry. The morphological data could be correlated with either tip speed or the "energy dissipation/circulation function," but a reasonable correlation of the specific growth rate and specific production rate was only possible with the latter. Copyright 1998 John Wiley & Sons, Inc.     

Model for Branch Initiation in Aspergillus nidulans Based on Measurements of Growth Parameters

The rate of hyphal elongation and the number of branches per hypha were measured on short sporelings of Aspergillus nidulans growing at different rates. The rate of elongation was proportional to total length in unbranched and branched hyphae. At each growth rate, the number of branches per hypha increased with increasing length and gave approximately straight-line graphs when plotted against length. The average number of branches per unit of hyphal length was quite different for the various growth rates and increased in direct proportion to the growth rate. The results are interpreted to mean that (i) growing tips have a maximum rate at which they can elongate and which is reached at hyphal lengths characteristic of the particular growth rate and (ii) a new branch is formed when the capacity of the hypha to elongate exceeds that of the existing tips.     

A quantitative approach to characterizing cell lysis caused by mechanical agitation of Streptomyces clavuligerus

Streptomyces clavuligerus is a commercially important actinomycete that is used to produce clavulanic acid, a beta-lactamase inhibitor. Observations during 10 batch cultivations with S. clavuligerus on defined media have led to the finding that the organism is very sensitive to shear when grown in batch cultures with increasing stirrer speed. The stirrer speed was increased to keep the dissolved oxygen level above 50% air saturation. A quantitative approach based on the calculation of elemental balances and a simple mathematical model is proposed to characterize the biomass lysis. Finally, a linear relation between biomass yield and observed specific growth rate is determined. Results show that cell lysis occurs at a high degradation rate, e.g., mu(max) = 0.16 h(-1) and k(d) = 0.07 h(-1), when the gassed power input increases above 1.1, 1.7, or 2.0 kW/m(3), respectively, depending on the medium composition. The overall biomass yield on substrate is dramatically reduced in all experiments (>30%).     

Effect of mixing rate on beta-carotene production and extraction by dunaliella salina in two-phase bioreactors

beta-Carotene has many applications in the food, cosmetic, and pharmaceutical industries; Dunaliella salina is currently the main source for natural beta-carotene. We have investigated the effect of mixing rate and whether it leads to the facilitated release of beta-carotene from the cells of Dunaliella salina in two-phase bioreactors. Three pairs of bioreactors were inoculated at the same time, operated at 100, 150, and 170 rounds per minute, respectively, and illuminated with a light intensity of 700 micromol m(-2) s(-1). Each pair consisted of one bioreactor containing only aqueous phase for the blank and one containing the water phase together with dodecane, which is biocompatible with the cells. Comparison of the viability and growth of the cells grown under different agitation rates shows that 170 rpm and 150 rpm are just as good as 100 rpm. The presence and absence of the organic phase also has no influence on the viability and growth of the cells. In contrast to the growth rate, the extraction rate of beta-carotene is influenced by the stirrer speed. The extraction rate increases at a higher stirring rate. The effectiveness of extraction with respect to power input is comparable for all the applied mixing rates, even though it is slightly lower for 100 rpm than the others. The chlorophyll concentration in the organic phase remained very low during the experiment, although at higher mixing rates, chlorophyll impurity increased up to 3% (w/w) of the total extracted pigments. At 170 rpm carotenoid and chlorophyll undergo the highest extraction rate for both pigments-0.5% of the chlorophyll and 6% of the carotenoid is extracted.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.     

The kinetics of yeast growth on pure hydrocarbons

The kinetics of C. tropicalis growth were investigated with pure n-hexadecane as dispersed phase substrate. Two distinct growth phases were found: In the first phase, exponential growth was independent of stirrer speed. The onset of the second phase, one of linear growth, was determined by stirrer speed. By the use of two different fermenter types, it was shown that the drop size of the dispersed phase was not primarily responsible for the observed kinetics. It was considered that the formation of biological flocs determined the observed growth pattern. This was substantiated by the results of continuous cultures in the different fermenter types, with various substrate concentrations.     

Performance test of a 6-stage continuous reactor for palm methyl ester production

Effects of residence time (3-12 min), stirrer speed (0-800 rpm), and NaOH concentration (0.25-1.0 wt% of oil) on the production performance of the designed 6-stage continuous reactor (2.272 l) for transesterification of palm oil were investigated at molar ratio of methanol to oil of 6:1 and temperature of 60 degrees C. Higher stirrer speed increased the reaction rate up to an appropriate speed but excessive stirrer speed decreased the reaction rate. Inappropriate stirrer speed runs dramatically decreased the production capacity of the reactor. Higher NaOH concentration significantly increased reaction rate and production capacity of the reactor. The reactor had a residence time distribution equivalent to 5.98 ideal CSTRs in series and a production performance equivalent to a plug flow reactor. At NaOH of 1.0 wt% of oil, the reactor could produce saleable biodiesel within residence time of 6 min in which a production capacity was 17.3 l/h and a power consumption of stirrer was 0.6 kW/m(3).     

The effect of culture conditions on the production of erythromycin by Saccharopolyspora erythraea in batch culture

Summary Saccharopolyspora erythraea growth is inhibited when grown at a low constant dissolved oxygen tension (DOT) of 10% air saturation. However, the specific erythromycin production is virtually identical to that of a culture where the DOT did not fall below 65%. In addition, at constant DOT (10%) a stirrer speed of 750 rpm in a 7 litre causes mechanical damage to the mycelia in comparison with result at 500 rpm.