Advances in shaking technologies

Shaking bioreactors are the most frequently used reactor system for screening and process optimization on a small scale. Their success can be attributed to their simple and functional design, which make shaking systems suitable for a large number of cost-efficient parallel experiments. Recently reported findings for oxygen transfer, power input, out-of-phase operation, hydromechanical stress and mixing in shaken bioreactors are summarized in this article. Novel monitoring techniques for the control of culture conditions in shake flasks and microtiter plates are described. The methods for characterizing culture conditions and the novel online measurement techniques that are summarized in this article can be utilized to tap the full potential of shaking reactor systems.     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Production of swainsonine from Metarhizium anisopliaein stirred-tank and air-lift reactors

Growth of Metarhizium anisopliaein modified starch-casein medium produced 61, 50, and 58 mg swainsonine/l when cultured in shaken flasks, stirred-tank and air-lift reactors respectively. Over approximately 45 h, the maximum swain-sonine specific productivity was 0.47 mg/g.h in a stirred tank reactor and 0.32 mg/g.h in an air-lift reactor. After 120 h, increasing broth viscosity was encountered in the latter fermenter.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Reaction engineering analysis of cellulase production with Trichoderma reesei RUT-C30 with intermittent substrate supply

The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L^?1 microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L^?1 day^?1 intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL^?1 with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL^?1).     

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.     

Miniature bioreactors: current practices and future opportunities

This review focuses on the emerging field of miniature bioreactors (MBRs), and examines the way in which they are used to speed up many areas of bioprocessing. MBRs aim to achieve this acceleration as a result of their inherent high-throughput capability, which results from their ability to perform many cell cultivations in parallel. There are several applications for MBRs, ranging from media development and strain improvement to process optimisation. The potential of MBRs for use in these applications will be explained in detail in this review. MBRs are currently based on several existing bioreactor platforms such as shaken devices, stirred-tank reactors and bubble columns. This review will present the advantages and disadvantages of each design together with an appraisal of prototype and commercialised devices developed for parallel operation. Finally we will discuss how MBRs can be used in conjunction with automated robotic systems and other miniature process units to deliver a fully-integrated, high-throughput (HT) solution for cell cultivation process development.     

The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations

Most experiments in screening and process development are performed in shaken bioreactors. Today, microtiter plates are the preferred vessels for small‐scale microbial cultivations in high throughput, even though they have never been optimized for this purpose. To interpret the experimental results correctly and to obtain a base for a meaningful scale‐up, sufficient oxygen supply to the culture liquid is crucial. For shaken bioreactors this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling and the well geometry affect the maximum oxygen transfer capacity (OTR_max) in microtiter plates. On a 48‐well plate scale, 30 different cross‐section geometries of a well were studied. It could be shown that the introduction of baffles into the common circular cylinder of a microtiter plate well doubles the maximum oxygen transfer capacity, resulting in values above 100 mmol/L/h (k_La > 600 1/h). To also guarantee a high volume for microbial cultivation, it is important to maximize the filling volume, applicable during orbital shaking. Additionally, the liquid height at the well bottom was examined, which is a decisive parameter for online‐monitoring systems such as the BioLector. This technology performs fiber‐optical measurements through the well bottom, therefore requires a constant liquid height at all shaking frequencies. Ultimately, a six‐petal flower‐shaped well geometry was shown to be the optimal solution taking into account all aforementioned criteria. With its favorable culture conditions and the possibility for unrestricted online monitoring, this novel microtiter plate is an efficient tool to gain meaningful results for interpreting and scaling‐up experiments in clone screening and bioprocess development. Biotechnol. Bioeng. 2009;103: 1118–1128. © 2009 Wiley Periodicals, Inc.     

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

Glucose-containing polymer rings enable fed-batch operation in microtiter plates with parallel online measurement of scattered light,fluorescence, dissolved oxygen tension,and pH

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.     

Enhancement of Cephalosporin C production by cultivation of Cephalosporium acremonium M25 using a mixture of inocula

AIMS: To enhance the productivity of Cephalosporin C (CPC) by cultivation of Cephalosporium acremonium M25 using a mixture of inocula. METHODS AND RESULTS: Inoculum age was classified into three stages (early, intermediate and late) by image analysis. A mixture of inocula, according to the inoculum ages, was used for efficient production of CPC in the main culture. The most effective mixing ratio of inocula for CPC production in shake flasks was a 3 : 7 volume ratio of early- and late-stage inocula. This was also the case in a 1.5 l stirred-tank reactor. CPC productivity was enhanced by about 32% and 34% when using an inoculum mixture in the shake flask and 1.5 l stirred-tank reactor, respectively. CONCLUSION: The morphological characteristics of C. acremonium M25 in the seed culture were quite different according to inoculum age. The compromise of different ages of inoculum showed better production of CPC. Significance and Impact of the Study: The productivity of CPC was enhanced considerably when using mixed inocula. The results of this study can be applied to fungal cultures for efficient production of various metabolites.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

Mini-scale bioprocessing systems for highly parallel animal cell cultures

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.     

Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non‐parallel and non‐equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag‐phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed‐batch mode in precultures in microtiter plates and shake flasks. Fed‐batch operation in precultures is realized through a slow‐release system for glucose. After differently growing cultures turn to glucose‐limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow‐release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high‐throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha. Biotechnol. Bioeng. 2009;103: 1095–1102. © 2009 Wiley Periodicals, Inc.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).