Shear conditions in clavulanic acid production by Streptomyces clavuligerus in stirred tank and airlift bioreactors

In biochemical processes involving filamentous microorganisms, the high shear rate may damage suspended cells leading to viability loss and cell disruption. In this work, the influence of the shear conditions in clavulanic acid (CA) production by Streptomyces clavuligerus was evaluated in a 4-dm(3) conventional stirred tank (STB) and in 6-dm(3) concentric-tube airlift (ALB) bioreactors. Batch cultivations were performed in a STB at 600 and 800 rpm and 0.5 vvm (cultivations B1 and B2) and in ALB at 3.0 and 4.1 vvm (cultivations A1 and A2) to define two initial oxygen transfer conditions in both bioreactors. The average shear rate ([Formula: see text]) of the cultivations was estimated using correlations of recent literature based on experimental data of rheological properties of the broth (consistency index, K, and flow index, n) and operating conditions, impeller speed (N) for STB and superficial gas velocity in the riser (UGR) for ALB. In the same oxygen transfer condition, the [Formula: see text] values for ALB were higher than those obtained in STB. The maximum [Formula: see text] presented a strong correlation with a maximum consistency index (K (max)) of the broth. Close values of maximum CA production were obtained in cultivations A1 and A2 (454 and 442 mg L(-1)) with similar maximum [Formula: see text] values of 4,247 and 4,225 s(-1). In cultivations B1 and B2, the maximum CA production of 269 and 402 mg L(-1) were reached with a maximum [Formula: see text] of 904 and 1,786 s(-1). The results show that high values of average shear rate increase the CA production regardless of the oxygen transfer condition and bioreactor model.     

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.     

An engineering analysis of rotating sieves for hybridoma cell retention ins stirred tank bioreactors

The use of internal rotating sieves for perfused hybridoma culture offers unique advantages but has been up to now largely empirical. Calculations have been performed on a 15 l spinfilter stirred tank in order to have an idea of hydrodynamic conditions inside and outside the rotating sieve. The large peripheral velocity value, resulting from sieve rotation (compared to axial and radial velocities) is expected to affect strongly sieve surface colonization by cells; this is confirmed by lab scale experiments, showing that cell colonization is prevented providing sieve rotation exceeds a defined value (around 0.6 m.s.1 tip speed); the fluid removal force calculated under these conditions appears to be in the range of 10 pN, similar to the adhesion force already reported for mammalian cells attached to inorganic substrata.     

Prediction of gas-liquid mass transfer coefficient in sparged stirred tank bioreactors

Oxygen mass transfer in sparged stirred tank bioreactors has been studied. The rate of oxygen mass transfer into a culture in a bioreactor is affected by operational conditions and geometrical parameters as well as the physicochemical properties of the medium (nutrients, substances excreted by the micro-organism, and surface active agents that are often added to the medium) and the presence of the micro-organism. Thus, oxygen mass transfer coefficient values in fermentation broths often differ substantially from values estimated for simple aqueous solutions. The influence of liquid phase physicochemical properties on kLa must be divided into the influence on k(L) and a, because they are affected in different ways. The presence of micro-organisms (cells, bacteria, or yeasts) can affect the mass transfer rate, and thus kLa values, due to the consumption of oxygen for both cell growth and metabolite production. In this work, theoretical equations for kLa prediction, developed for sparged and stirred tanks, taking into account the possible oxygen mass transfer enhancement due to the consumption by biochemical reactions, are proposed. The estimation of kLa is carried out taking into account a strong increase of viscosity broth, changes in surface tension and different oxygen uptake rates (OURs), and the biological enhancement factor, E, is also estimated. These different operational conditions and changes in several variables are performed using different systems and cultures (xanthan aqueous solutions, xanthan production cultures by Xanthomonas campestris, sophorolipids production by Candida bombicola, etc.). Experimental and theoretical results are presented and compared, with very good results.     

High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production

Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.     

β - glucosidase production by Aspergillus wentii in stirred tank bioreactors

Summary Batch cultivation of Aspergillus wentii, Wehmer, Pt 2804 was carried out on Mandels and Reese (1957) medium containing 3% cellulose and 0.3% peptone at 30°C in 14 and 30 liter Bioreactors. Use of a 48 hours old inoculum (10% v/v), 0.5 vvm aeration at 400–800 rpm and maintenance of dissolved oxygen and pH above 50% saturation level and between 5.2 to 4.0 respectively, produced 10.58 g cells/lit and 61.9 IU/1/hr cellobiase productivity.     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors

Miniature bioreactors under parallel fed‐batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale‐up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed‐batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred‐tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed‐batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD₆₀₀) of 114–133 and final DCW concentrations of 69–70 g L^–1. The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.     

High density and scale-up cultivation of recombinant CHO cell line and hybridomas with porous microcarrier Cytopore

Using porous microcarrier Cytopore and a low-serum medium supplement BIGBEF-3, we have successfully cultivated recombinant CHO cell line CL-11G producing prourokinase and hybridomas producing anti-prourokinase monoclonal antibody in Celligen 1.5 or 5 L bioreactor. The cell density obtained ranged from 1 to 2 × 107 cells mL-1. The yields of prourokinase and monoclonal antibody increased with increasing cell density. As the cells could spontaneously release from and reattach to porous microcarriers, it was very easy to scale-up the cultivation. Thus the bead to bead cell transfer method has been used to scale up the cultivation of CL-11G cells to a 20 L reactor-scale for the pilot production of prourokinase, and also to scale-up the culture of hybridomas for the production of monoclonal antibody for the purification of prourokinase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.     

Biodegradation of phenol in a continuous process: comparative study of stirred tank and fluidized-bed bioreactors

The paper presents the main results obtained from the study of the biodegradation process of phenol by a pure culture of Pseudomonas putida ATCC 17484. The experimental work was carried out in two different systems: a stirred tank where cells grew as a suspended culture and a fluidized bed where cells were immobilized within calcium alginate gel beads. The influence of the hydraulic residence time (HRT) and organic loading rate on the removal efficiency of phenol was determined for both bioreactors. Also, the stability of the fluidized-bed bioreactor (FBB) in terms of its ability to withstand sudden phenol overdoses is also reported. Experimental values indicated that both bioreactors showed high phenol degradation efficiencies, higher than 90%, even for a phenol loading rate in the influent as high as 4 g phenol/l day. The FBB showed better performance than the suspended-culture bioreactor due to its better control and because it could operate with lower HRT.     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

Comparisons of optically monitored small-scale stirred tank vessels to optically controlled disposable bag bioreactors

Background  

Upstream bioprocesses are extremely complex since living organisms are used to generate active pharmaceutical ingredients (APIs). Cells in culture behave uniquely in response to their environment, thus culture conditions must be precisely defined and controlled in order for productivity and product quality to be reproducible. Thus, development culturing platforms are needed where many experiments can be carried out at once and pertinent scale-up information can be obtained.     

Comparison of stirred tank and airlift bioreactors in the production of polygalacturonases by Aspergillus oryzae

The production of endo and exo-polygalacturonase (PG) by Aspergillus oryzae IPT 301 was studied in a stirred tank bioreactor (STR) and an internal circulation airlift bioreactor. Using a factorial experimental design, a soluble culture medium was defined which allowed the production of exo- and endo-PG comparable to that obtained in a medium containing suspended wheat bran. The soluble medium was used in tests to compare the production of these enzymes in the STR and airlift bioreactor. In these tests, after 96 h, maximum enzymatic activity values achieved for exo- and endo-PG were 65.2 units (U) per mL and 91.3 U mL⁻¹, in the STR, with similar activity values of 60.6 U mL⁻¹ and 86.2 U mL⁻¹, respectively, being achieved in the airlift bioreactor. The airlift bioreactor also showed satisfactory results regarding the oxygen transfer rate in this process, indicating its potential to be used in an eventual larger scale production of exo- and endo-PG, with lower costs for both installation and operation.     

Immobilized mammalian cell cultivation in hollow fiber bioreactors

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.