Oxygen transfer efficiency in the biosynthesis of antibiotics in bioreactors with a modified RUSHTON turbine agitator

In this work, the oxygen mass transfer efficiency and power consumption in a non-biological system and an antibiotic biosynthesis process, using a modified RUSHTON turbine agitator, were investigated. It was demonstrated that a simple modification of the blades through the increase of the blade height, simultaneously with the discontinuation of the blade surface, could improve the oxygen transfer efficiency by about 30%. Experiments performed in stirred tank bioreactors with an overall volume of 20 m³, equipped with the modified RUSHTON turbine agitator, showed that the power consumption diminished by a factor of 1.18 to 1.6 during the fermentation processes of Streptomyces erithreus, Streptomyces griseus, Streptomyces noursei, and Nocardia mediaterranei, compared to the witness bioreactor. The use of the modified RUSHTON turbine for the antibiotic biosynthesis process may contribute to the decrease of the overall costs and the obtainment of better productivity, allowing an intensive utilization of power inputs for aeration and agitation.     

Studies on transfer processes in mixing vessels: effect of gas on solid-liquid hydrodynamics using modified Rushton turbine agitators

The effect of gas on solid-liquid hydrodynamics in mixing vessels was studied to determine the agitation speed required to just completely suspend all the particles under gassed conditions, N _jsg and by measurement of the power consumption associated with this agitation speed. The solid particles have a mean diameter between 15–1000 μm. For their mixing are used standard and modified Rushton turbine agitators positioned singly or doubly on the same shaft. The modified turbine with a surface fraction of the perforations equal to 0.353 (TP3) was obtained through increase in the blade height of the Rushton turbine simultaneously with the perforation of the blade surface. The filled surface of the modified blade is equal to the blade surface of the standard Rushton turbine.     

Studies on transfer processes in mixing vessels: effect of particles on gas-liquid hydrodynamics using modified Rushton turbine agitators

The work presents the effect of solid particles having a mean diameter between 15–1000?μm, on the gas dispersion in a mechanically agitated vessel with standard and modified Rushton turbine agitators positioned singly or doubly on same shaft. For the dispersing and uniform distribution of the three phase (gas-liquid-solid) through the entire vessel section, the modified blade turbines, with the surface fraction of the perforations equal to 0.353, were found to be more efficient, the power consumption being reduced by approximately 50%in comparison with the standard Rushton turbines. The power number in the turbulent mixing of the three phase system is dependent on the aeration rate, the surface fraction of the perforations, the turbine number and the physical and rheological properties of the suspensions.     

Rheological behaviour of some antibiotic biosynthesis liquids

This paper prsents the results of teh study of rheological behaviour of antibiotic biosynthesis liquids obtained by submerged aerobic cultivation of microorganisms belonging to the actinomycete and fungi classes, in stirred tank bioreactors with turbine impellers. These liquids have a non-Newtonian behaviour which follows the power-law rhcological model with a correlation index of over 0.95. The studied liquids are pseudoplastic, and alter their rheological properties, such as consistency index, (K), flow index, (n), apparent viscosity, (η_a), maximum Newtonian viscosity (η₀), with the culture age, microrganism strain and batch conditions. Also, these liquids are time dependent, exhibiting thixotropy. The most viscous liquids are produced by Streptomyces aureofaciens and Streptomyces rimosus cultivation, while that produced by Streptomyces griseus is the least viscous. A higher pseudoplasticity appears after 30 hours culture age. Since all these biosynthesis are aerobic, a careful observation of the rhelogical behaviour dynamics is necessary to avoid the oxygen culture supply limitation and the decrease of the bioreactor performance during biosynthesis.     

Studies on transfer processes in mixing vessels: suspending of solid particles in liquid by modified Rushton turbine agitators

The modified blade turbines are attractive alternatives to the standard Rushton turbine as they do not require any modification in the electrical engine motor and drive assemblies are simple to manufacture and have a reduced power consumption.The modified blades were obtained through increase in the blade height of the Rushton turbine simultaneously with perforation of the blade surface. The field surface of the modified blade is equal to the blade surface of the standard Rushton turbine.In this study the modified blade turbine with the surface fraction of the perforations equal to 0.353 is used.The complete suspension speed and the power dissipation in transition and turbulent regimes using standard and modified Rushton turbine agitators positioned singly or doubly on same shaft, in five solid-liquid systems were investigated.The solid particles used have the mean diameter between 15–1000 m.The modified blade turbine, noted as TP3, was found to be more efficient than the standard turbine in complete and homogeneous suspension.List of Symbols A distance between turbine and the vessel bottom (m) - c dimensionless constant (-) - d agitator diameter (m) - d _p surface-to-volume mean diameter of the particle (m) - D vessel diameter (m) - (H _L )₁ suspension height for one turbine immersed (m) - (H _L )₂ suspension height for two turbines immersed (m) - K consistency index (Pa s ⁿ ) - l _k eddy-size characteristic (m) - N flow behaviour index (-) - N _p number of blades of the mixing system (-) - N agitator speed (s^–1) - N _js agitator speed that just causes complete suspension (s^–1) - Ne P_L/_LN³d⁵ power number in liquid system (-) - (Ne) _g P_g/_spN³d⁵ power number in solid-liquid system (-) - P _L power consumption in liquid system (W) - P _s power consumption in solid-liquid system (W) - r coefficient of correlation (-) - R distance between turbines (m) - Re _spNd²/ _a Reynolds number (-) - S suspension parameter in Zwietering equation (2) (-) - S _C full surface of the blade (m²) - S _G surface of the perforations applied on the blade (m²) - S _G /S _C surface fraction of the perforations (-) - X particle concentration (g/l) - w baffle width (m) - _js specific power input per mass at the complete suspension state (W/kg) - _a apparent viscosity under mixing conditions (Pa s) - _L kinematic viscosity of the liquid (m²/s) - _L density of liquid (Kg/m³) - _s density of solid (Kg/m³) - _sp density of suspension (Kg/m³)     

Studies on the production of lipase from recombinant Staphylococcus carnosus

Summary Production of lipase from recombinant Staphylococcus carnosus pLipPS2 was studied in standard stirred tank bioreactors. Only low lipase activity was obtained under conventional operating conditions, i.e., moderate to high stirring speeds and aeration rates for keeping the dissolved oxygen concentration at high levels. Additional targetted experiments indicated that the reason for the observed low lipase activity is lipase inactivation due to surface forces and shear stress at the gas/liquid interface. Therefore, a cultivation strategy is proposed that minimizes gas/liquid interfacial area and maximizes the driving concentration for O₂ mass transfer by controlling the dissolved oxygen to low values by gentle stirring and low aeration rates. Thus, high lipase activities can be obtained even in larger scale standard stirred tank bioreactors. Offprint requests to: W.-D. Deckwer     

The influence of impeller type in pilot scale xanthan fermentations

The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.     

Zur Maßstabsübertragung von aeroben mikrobiologischen Prozessen Teil I. Stofftransport

Because the interior scale of turbulence in reinforced bioreactors is essentially greater than the particles of bacteria or yeasts, the mass transfer occurs at the particles only in consequence of the molecular diffusion and depends on the concentration of the soluted material only. As a decided criterion for the transmission of scale in such processes is considered the volumetric oxygen transfer coefficient K_La This article practises a theoretical analysis of the characteristic hydrodynamic conditions in bubble columns and in agitators: the content of gas, the specific area of mass transfer and the coefficient of mass transfer K_L. These conditions are joined with the evaluation of the K_La value. Resultant the data of calculation hence it follows a computational determination of the K_La value by means of the physical matter values, the construction of devises and the process conditions.     

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of ± pH 0.2 if the set-point is pH 6.0 or lower.     

Scale-up on basis of structured mixing models: A new concept

A new scale-up concept based upon mixing models for bioreactors equipped with Rushton turbines using the tanks-in-series concept is presented. The physical mixing model includes four adjustable parameters, i.e., radial and axial circulation time, number of ideally mixed elements in one cascade, and the volume of the ideally mixed turbine region. The values of the model parameters were adjusted with the application of a modified Monte-Carlo optimization method, which fitted the simulated response function to the experimental curve. The number of cascade elements turned out to be constant (N = 4). The model parameter radial circulation time is in good agreement with the one obtained by the pumping capacity. In case of remaining parameters a first or second order formal equation was developed, including four operational parameters (stirring and aeration intensity, scale, viscosity). This concept can be extended to several other types of bioreactors as well, and it seems to be a suitable tool to compare the bioprocess performance of different types of bioreactors. (c) 1994 John Wiley & Sons, Inc.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

On the pollen of Salpianthus (Nyctaginaceae)

Pollen grains of the three recognized species of Salpianthus (Nyctaginaceae), i.e., S. arenarius Humb. & Bonpl., S. macrodontus Standl., and S. purpurascens (Cav. Ex Lag.) Hook &. Arn. were studied with special reference to the presence and number of spinules and perforations per area unit, size, shape and polar area index. Image analysis of SEM‐micrographs at a standard magnification of χ 15,000 and in a specific area of 6 χ 6 cm was performed by means of Leica Quantimet 570. Counts were also made manually and the two compared statistically. Shape, polar area index, and size were calculated by different statistical tests. S. macrodontus has larger pollen grains than S. arenarius and S. purpurascens; S. arenarius is more subprolate than the other two while S. purpurascens has a larger polar area index than S. arenarius and S. macrodontus. The number of spinules and perforations could not be used to separate the three species as intraspecific variation is greater than interspecific variation. No correlation between pollen morphology and leaf shape could be proved.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50‐mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high‐throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three‐dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k_La) was determined experimentally and from simulations under various working conditions. We also determined the liquid‐phase mass transfer coefficient (k_L) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k_L. High oxygen transfer rates, sufficient for supporting the high‐density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192–200, 2017     

Vessel dimensions in liana and tree species of Gnetum (Gnetales)

Maximum vessel diameters were examined in the secondary xylem of stems of Gnetum of various sizes. One tree (G. gnemon) and 13 liana species were compared. In three species, vessel length distributions were determined by the latex paint method, and showed many short and fewer long vessels. Latex and compressed air methods, used to find the maximum vessel lengths, showed that maximum vessel lengths were similar for three species of Gnetum. In old stems, mean and maximum vessel diameters tended to be greater in lianas than in the tree species. The skewed distribution of vessel lengths and the trend of wider vessels in lianas as compared to trees were similar to those distributions and trends described previously for angiosperms. In random samples of macerated wood of three species, simple perforation plates were most common in vessel members of all species. Foraminate and modified foraminate perforations were less frequent. Average diameter of vessel members with either foraminate or modified foraminate perforations was less than for those with simple perforations. The resemblance of Gnetum vessels to those of angiosperm trees and vines is most likely a case of convergent evolution (homoplasy) in xylem characteristics.     

Enhancement of mass transfer characteristics and phenanthrene degradation in a two-phase partitioning bioreactor equipped with internal static mixers

Oxygen and substrate supply have always been considered physical constraints for the performance and operation of two-phase partitioning bioreactors (TPPB), widely used for the degradation of hydrophobic substrates. In this regard, the potential advantages of static mixers in upgrading the oxygen transfer and liquid-liquid dispersions in TPPB have been highlighted. In the present paper, the concomitant influence of static mixers on the gas-liquid mass transfer coefficient k _L a and on substrate bioavailability was examined in TPPB. The static method based on conventional forms was developed to estimate the oxygen volumetric mass transfer coefficient. Over a broad range of liquid and air flow rates, the presence of static mixers was found to significantly enhance k _L a relative to a mixer-free mode of operation. For identical conditions, static mixers improved the k _L a threefold. In the presence of external aeration supply, the boost in the k _L a was associated with an increase of 16% in the phenanthrene biodegradation rate due to bubble break up accomplished by the static mixers. On the other hand, static mixers were efficient in enhancing substrate bioavailability by improving the liquid-liquid interfacial area. This effect was reflected by a threefold increase in the degradation rate in the bioreactors with no external supply of air when equipped with static mixers.     

Methods and milliliter scale devices for high-throughput bioprocess design

Based on electromagnetic simulations as well as on computational fluid dynamics simulations gas-inducing impellers and their magnetic inductive drive were optimized for stirred-tank reactors on a 10 ml-scale arranged in a bioreaction block with 48 bioreactors. High impeller speeds of up to 4,000 rpm were achieved at very small electrical power inputs (63 W with 48 bioreactors). The maxima of local energy dissipation in the reaction medium were estimated to be up to 50 W L⁻¹ at 2,800 rpm. Total power input and local energy dissipation are thus well comparable to standard stirred-tank bioreactors. A prototype fluorescence reader for 8 bioreactors with immobilized fluorometric sensor spots was applied for online measurement of dissolved oxygen concentration making use of the phase detection method. A self-optimizing scheduling software was developed for parallel control of 48 bioreactors with a liquid-handling system for automation of titration and sampling. It was shown on the examples of simple parallel batch cultivations of Escherichia coli with different media compositions that high cell densities of up to 16.5 g L⁻¹ dry cell mass can be achieved without pH-control within 5 h with a high parallel reproducibility (standard deviation<3.5%, n=48) due to the high oxygen transfer capability of the gas-inducing stirred-tank bioreactors.     

Development of a method for reliable power input measurements in conventional and single‐use stirred bioreactors at laboratory scale

Power input is an important engineering and scale‐up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory‐scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single‐use bioreactors in the single‐digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of the measurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (N_P = 4.17 ± 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single‐use bioreactors could be determined over a wide range of Reynolds numbers between 100 and >10⁴. Power numbers of between 0.3 and 4.5 (for Re = 10⁴) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque‐based technique can be used to reliably measure power input in stirred reusable and single‐use bioreactors at the laboratory scale.