Shake Bioreactor Research Articles

Determination of CO2 sensitivity of micro‐organisms in shaken bioreactors. I. Novel method based on the resistance of sterile closure

Influence of carbon dioxide on growth and product kinetics of industrially important micro-organisms is essential for the interpretation of a bioprocess. In this research, the CO₂ effects on productivity and growth rate of micro-organisms have been studied by using a variety of kplug. The applied method is based on a different concentration of CO₂ in the headspace of ventilation flasks. The presented method is simple, inexpensive and shows similar results compared to large-scale fermentation regarding the evolution of CO₂ in a batch system. For the investigation of the proposed method, experiments employing Arxula adeninivorans LS3, Corynebacterium glutamicum (DM1730 and ATCC WT13032) and Hansenula polymorpha DSM70277 as model organisms in the ventilation flasks are performed. The fermentations in the RAMOS (respiratory activity monitoring system) device were carried out with a normal aeration rate (1 vvm) and under the same operating conditions as the ventilation flask f1. The modified unsteady-state model was used to predict the operation conditions of a biological system in the ventilation flasks. In the present study, a novel and easy method for the quantification of CO₂ sensitivity of micro-organisms in shaken bioreactors (called ventilation flask) was achieved.

Transient Expression of Recombinant sPDGFRα-Fc in CHO DG44 Cells using 50-mL Orbitally Shaking Disposable Bioreactors

Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFR alpha-Fc fusion protein expressed in static CHO-k(1) cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFR alpha-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFR alpha-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFR alpha-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expression in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies.

SSPRSD Using a Filamentous Fungal Strain Penicillium expansum BS30 Isolated from Wastewater Sludge

A filamentous fungal strain (Penicillium expansum BS30) isolated from a municipal wastewater treatment plant was used in this study to simultaneously reduce sludge solids, pathogens, and improve the sludge settling and dewaterability [simultaneous solids and pathogens reduction, settling and dewatering (SSPRSD)] in shake flask and 10-L bioreactor experiments. The fungal strain role in the SSPRSD process was evaluated at different temperatures and inoculum (spores) concentrations. The best performance of the process was achieved at incubation temperature of 25°C and inoculum concentration of 106 spores/mL . At these optimal conditions, suspended solids (SS) and volatile SS were degraded >50 and >53% , respectively. The capillary suction time value recorded ( <13 s ) was lower than that required for sludge dewaterability ( <20 s ) . The populations of total coliforms and Salmonella (pathogen indicators) were reduced by two and four log cycles, respectively. A study on molecular screening of penicillin bios...

Comparative studies and identification of camptothecin produced by an endophyte at shake flask and bioreactor

The fungus showing homology with Nodulisporium by 28S ribosomal gene sequencing, which has been discovered as an endophyte on medicinal plant Nothapodytes foetida, was found to produce 45 and 5.5 µg of camptothecin (CPT) per gram of mycelia at bioreactor and at shake flask, respectively, which was further quantified and characterised by various spectroscopic analyses.

Biotechnological approaches for the production of guggulsterones through organised and unorganised cultures of Commiphora wightii in shake flask and bioreactor

Biotechnological approaches for the production of guggulsterones through organised and unorganised cultures of Commiphora wightii in shake flask and bioreactor

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.

Enhanced Human Lysozyme Production by Kluyveromyces lactis

An attempt to enhance recombinant human lysozyme production by Kluyveromyces lactis K7 was performed in this study. In this study, the production of recombinant human lysozyme was investigated using shake flasks and bioreactor under different cultivation conditions. It was demonstrated that 25 °C could enhance human lysozyme production when compared with other temperatures tested. This study also demonstrated that higher biomass did not necessarily produce higher human lysozyme, and it was clear that human lysozyme production was enhanced under unfavorable conditions such as low acidity and oxygen limitation. Cultivation condition with no pH control demonstrated better production than with pH controlled near neutrality. Oxygen limitation also resulted in higher recombinant human lysozyme production. Overall human lysozyme was increased from 64.1 U/ml in flask to 123.6 U/ml in fed-batch fermentation using 4.5% (w/v) glucose initially and fed with concentrated lactose to achieve 9% lactose concentration.

Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633

The effect of oxygen transfer rate (OTR) on the synthesis of mycosubtilin, a non ribosomal lipopeptide antifungal biosurfactant, was investigated in the respiration activity monitoring system (RAMOS) for two Bacillus subtilis strains. These cultures were performed under definite oxygen-limited conditions without the adding of any anti-foam in the culture medium. By using four different filling volumes (FV) in the shaken bioreactors, different levels (20, 14, 9 and 7 mmol O 2 l −1 h −1) of oxygen-limited growth could be obtained. A 25-fold increase of the specific productivity of mycosubtilin was observed for B. subtilis ATCC6633 in the case of the most severe oxygen limitation. But nearly no effect could be found with strain BBG100 carrying the constitutive P repU promoter instead of the natural P myc promoter. Transcript analysis of the fenF gene belonging to the myc operon indicated that the P myc promoter regulation could be slightly oxygen sensitive. Additionally, different patterns of the synthetised mycosubtilin homologues were obtained for different level of oxygen-limited growths. At the present state of investigation, oxygen regulation was thus shown to act at different levels suggesting the existence of a complex regulatory system of NRPS lipopeptide synthesis in the natural B. subtilis ATCC6633 strain.

Extended Method to Evaluate Power Consumption in Large Disposable Shaking Bioreactors

Power consumption in large disposable shaking bioreactors can be readily measured by monitoring the temperature change of a fluid over time until steady state. However, as viscosity changes with temperature and power consumption depends on viscosity, no defined conditions exist. Therefore, an extended temperature method for determination of power consumption in disposable large shaking bioreactors of size >2 L is introduced and compared with the conventional temperature method. Dynamic simultaneous parameter estimation using several different data sets is performed for evaluation of model parameters. Results indicate suitability of the new method for measurement of power consumption for fluids possessing water-like viscosity as well as for viscous Newtonian fluids. The maximum values of power consumption obtained for water and for an 80% glycerol/water system is approximately 10 kW/m3 and 23 kW/m3, respectively. A non dimensional description of power consumption is given. The unusual behaviour observed in the value of power consumption in the 50 L vessel reveals that, under specific conditions, obviously “out-of-phase” conditions are encountered, necessitating further investigation at different shaking diameters.

Novel Orbital Shake Bioreactors for Transient Production of CHO Derived IgGs

Large-scale transient gene expression in mammalian cells is being developed for the rapid production of recombinant proteins for biochemical and preclinical studies. Here, the scalability of transient production of a recombinant human antibody in Chinese hamster ovary (CHO) cells was demonstrated in orbitally shaken disposable bioreactors at scales from 50 mL to 50 L. First, a small-scale multiparameter approach was developed to optimize the poly(ethylenimine)-mediated transfection in 50 mL shake tubes. This study confirmed the benefit, both in terms of extended cell culture viability and increased product yield, of mild hypothermic cultivation conditions for transient gene expression in CHO cells. Second, the scalability of the process was demonstrated in disposable shake bioreactors having nominal volumes of 5, 20, and 50 L with final antibody yields between 30 and 60 mg L(-1). Thus, the combination of transient gene expression with disposable shake bioreactors allows for rapid and cost-effective production of recombinant proteins in CHO cells.

A Novel and Easy Method for Quantification of CO2‐Sensitivity of Microorganisms in Shaken Bioreactors

A Novel and Easy Method for Quantification of CO₂‐Sensitivity of Microorganisms in Shaken Bioreactors

Optimization of cell seeding efficiencies on a three-dimensional gelatin scaffold for bone tissue engineering.

Bone tissue engineering techniques hold great potential for the treatment of clinical defects. However, there is much optimization needed before bone tissue engineering can be used therapeutically. This study evaluated various cell seeding methods onto a porous three-dimensional (3D) scaffold for bone tissue engineering optimization. MG63 human osteoblast-like cells were seeded onto a resorbable, porous gelatin sponge in different suspension volumes (50 microl and 5 ml), and culture conditions, (static, shaken, rolled, or rotatory bioreactor). The DNA of the cells in the scaffold, the media and the containers were quantitated separately to determine the cell number and location after 3 days of culture. The samples were stained with calcein and viewed using confocal microscopy to determine cell viability and location. Placing a small cell suspension (50 microl) directly onto the scaffold produced a significantly higher proportion of cells adhered to the scaffold than a larger cell suspension (5 ml). In all conditions except the rotatory bioreactor, the percentage of cells remaining on the scaffold after 3 days in a small seeding volume (63 +/- 22%) was significantly higher than the larger seeding volume (36 +/- 25%). In the case of the rotatory bioreactor, the opposite appeared to be true (39 +/- 9% small volume and 72 +/- 14% larger volume). It was important to keep the seeding dynamics of the cultivated tissue engineered construct consistent throughout the experiments to ensure reproducibility. For this scaffold type, cells applied in a small volume and cultured on a plate shaker at 120 rpm (giving 81 +/- 14% of cells adhered to the scaffold) for 3 days is recommended.

Oxygen limitation is a pitfall during screening for industrial strains

Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k(L)a) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space-time yield is discussed.

Fluid mixing in shaken bioreactors: Implications for scale-up predictions from microlitre-scale microbial and mammalian cell cultures

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process ( 1000 μ l ) for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant k L a values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells ( 800 μ l ) to conical flasks (100 ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures.

Interesting physiological response of the osmophilic yeast Candida krusei to heat shock

The response of cellular physiology of Candida krusei to heat shock was studied in shake flasks and bioreactors. Heat shock treatment increased trehalose content (based on g/g dry cells) and decreased glycerol content of C. krusei cells. The optimal heat shock temperature, period and timing condition was 45 °C for 1 h starting at the 12th h of cultivation. With this heat shock, the cellular trehalose content was 2.0, 2.8 and 3.8-fold that of control at 12.3, 12.6 and 13 h, respectively, while the corresponding glycerol content was 0.8, 0.6 and 0.4-fold that of control. When the temperature was shifted back to a normal one (from 45 °C to 35 °C) after the heat shock, the content of both trehalose and glycerol also returned to a control level. A further study on periodic heat shock treatment (at 45 °C for 1 h each time) was performed at 12, 18 and 24 h of cultivation, respectively. It was confirmed that the trehalose content of heat shocked cells was much higher than that of control at the end of each 1 h heat shock cycle (i.e., at 13, 19 and 25 h of cultivation), while the corresponding glycerol content was much lower than that of control. The dynamic enzyme activities of glycerol-3-phophate dehydrogenase (ctGPD), mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD) and trehalose-6-phosphate synthase (T-6-P synthase) were also investigated during heat shock. Different from ctGPD, both mtGPD and T-6-P synthase showed much higher activities for the heat shocked cells compared to the control. The results suggest that heat shock may alter the metabolic flux by shifting the pathway from glycerol to trehalose synthesis in C. krusei cells.

Power consumption and heat transfer resistance in large rotary shaking vessels

The application of disposable equipment is a new trend in bioprocessing. Liu and Hong (C.M. Liu, L.N. Hong, Development of a Shaking Bioreactor System for Animal Cell Cultures, Biochem. Eng. J. 7 (2) (2001) 121–125) have shown that the cultivation of animal cell cultures in large plastic shaking vessels of up to 56 L volume resulted in good performance in terms of product concentration. In order to characterize this type of reactor from a chemical engineering view point, the power consumption of a large rotary shaking vessel (20 L) by a temperature method was measured. It was shown that heat insulation of the large vessel is not required to measure power consumption and, therefore, a simple experimental setup could be employed. The power consumption per unit volume in the large vessel was found to be in the same range (≤10 kW/m 3) as that in small shaking flasks (<2 L). The dependency of power consumption on shaking frequency was less pronounced than that reported in previous papers within the experimental conditions investigated. Since the gas side heat transfer coefficient was the limiting step of heat transfer through the vessel wall, the overall heat transfer coefficient could be estimated using the power consumption per unit volume and the liquid filling volume. The temperature difference between the liquid and the surrounding air in case of the non-insulated vessel was found to be in the range of 5 K.

Introduction to advantages and problems of shaken cultures

Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation.

Synthesis of Poly(β-hydroxybutyrate) in Simulated Microgravity: An Investigation of Aeration Profiles in Shake Flask and Bioreactor

The microbial strain Azotobacter vinelandii UWD was grown under conditions of simulated microgravity in the National Aeronautics and Space Administration (NASA) Bioreactor. Bacterial growth in simulated microgravity differed significantly from that observed in conventional shake flask experiments: Cells tended to grow in a cluster-like pattern and polymer production started immediately after exposing them to conditions of simulated microgravity, and no lag time was observed. It was imperative to differentiate between the effects derived from microgravity and those imposed by the altered oxygen supply in the bioreactor. Aeration conditions were studied in both reactor types and a gas supply profile was developed for the bioreactor. This supply profile allowed for similar amounts of dissolved oxygen in the bioreactor and the shake flask in the initial stage of the fermentation and, therefore, for an evaluation of the effects of microgravity on biopolyester-producing bacteria. Since the optical density that is conventionally used as a measure for the cell growth could not be used due to the cluster-like growth pattern of the cells, it was determined that bacterial growth behavior in the bioreactor can be monitored through glucose or oxygen consumption.

Morphological development and gibberellin production by different strains of Gibberella fujikuroi in shake flasks and bioreactor

Strain H-984 of G. fujikuroi grown for 38 h in a shake flask with medium containing 20 g glucose l−1, 3 g yeast extract l−1, 2.5 g NH4NO3 l−1, 0.5 g KH2PO4 l−1, 0.1 g MgSO4 l−1, 1 g CaCO3 l−1, and inoculated into a bioreactor with medium containing 60 g glucose l−1; 1 g NH4Cl l−1; 3 g KH2PO4 l−1 and 1.5 g MgSO4 l−1 produced 1100 mg gibberellic acid l−1.