Small-scale Bioreactor Research Articles

Integrated methodology to evaluate bioremediation potential of creosote-contaminated soils

Integrated methodology including chemical analyses as well as biological and ecotoxicological testing of soil was used to evaluate bioremediation potential of an aged, highly contaminated soil from a wood preservation plant. The soils contaminated with coal tar creosote originated from two sites (Site I: 8000 mg/kg and Site II: 133,800 mg/kg). A toxicity test-battery able to detect different effects using a variety of endpoints was developed and applied for the ecotoxicological assessment of creosote in soils: Azotobacter agile and Pseudomonas fluorescens dehydrogenase enzyme activity test, Vibrio fischeri bioluminescence test, Sinapis alba root and shoot elongation test and Folsomia candida mortality test. The biodegradation and its enhancement were investigated in small-scale bioreactors in short-term laboratory experiments. The joint evaluation of chemical, biological and ecotoxicological results made possible to compare different technologies, and investigate their applicability for remediation of soils contaminated with coal tar. The most sensitive direct contact tests that also correlated well with the creosote-reduction were Vibrio fischeri bioluminescence test, followed by Folsomia candida mortality test and Sinapis alba shoot elongation test.

Cost‐effective engineering of a small‐scale bioreactor

Several methods exist for increasing the scale of cell culture in the laboratory. While these methods provide significant increases in biomass, they are often prohibitively expensive for many laboratories. We have engineered a small-scale bioreactor with a novel means of introducing oxygen through the catalytic breakdown of hydrogen peroxide using a manganese oxide catalyst. We have also adapted and modified an existing assay for dissolved oxygen to be compatible with culture conditions. In this system we have been able to culture CHO cells at densities of up to 10(7) cells/mL without the use of automated feedback systems.

A membrane-based small-scale bioreactor for accelerated in vitro drug screenings with primary hepatocytes

The use of primary hepatocyte cultures is well established for the study of drug metabolism and drug-drug interactions. However, the application of these cells in hybrid artificial liver systems has been hampered by the gradual loss of differentiated function in vitro. Therefore, several hepatic culture systems have been developed to circumvent this problem. In the present study, a small-scale bioreactor with a gas-permeable membrane was constructed in which primary hepatocytes were cultured in a sandwich model. The cells could preserve their specific functions such as protein synthesis, detoxification, and biotransformation when cultured in the bioreactor. Compared to the conventional well cultures, the use of the mini bioreactor has the benefit of promoting oxygen-dependent processes such as urea synthesis and Phase I biotransformation and of allowing induction of Phase I enzymatic activities by drugs.

Production of a recombinantly expressed laminin fragment by HEK293-EBNA cells cultured in suspension in a dialysis-based bioreactor

Laminin 5 is a multifunctional extracellular matrix protein, which supports epithelial cell adhesion through multiple cell binding sites. For elaborate studies, a 35 kDa fragment localized at the C-terminal extremity of the molecule, the LG4/5 fragment was recombinantly expressed in mammalian HEK293-EBNA cells. As the production of the LG4/5 fragment by adherent cell monolayers was very low (<1 μg/ml), we used the commercially available small scale bioreactor system miniPERM™. The HEK293-EBNA transfectants were adapted to grow in suspension in defined medium containing low level of fetal calf serum and produced the recombinant LG4/5 protein with quality consistent with that produced in conventional static cell culture conditions. Cells grew without forming aggregates in the bioreactor and the resulting HEK293-EBNA-LG4/5 cell line was suitable for unlimited passages in the bioreactor. 2.5 × 10 5 cells/ml were cultured for 25 days to reach the maximal cell density of 1.6 × 10 7 cells/ml. The quantification of protein synthesis revealed that the highest level of 2.4 mg of recombinant LG4/5 protein was harvested when 10 7 transfected cells/ml were injected in the bioreactor and allowed to grow for 2 days. The mean daily recombinant LG4/5 fragment product yield of 1.2 mg of protein per minifermenter shows that cultivation of HEK293-EBNA transfectants in suspension is highly convenient for the production of recombinant laminin fragments.

Performance of membrane bioreactors used for the treatment of wastewater from the chemical and textile industries

Within the scope of the study, nine waste waters from the chemical and textile industries were treated in bench-scale (laboratory scale) and small-scale (pilot scale) membrane bioreactors. Depending on wastewater characteristics, the resulting performance varied significantly. It was observed that MBR effectiveness was determined primarily by the degree of biodegradability of the wastewater. In the course of several months of operation, no significant changes associated with the complete retention of the biomass by the membranes were observed. In some cases, it was possible to improve effluent quality by using smaller molecular separation sizes. The flux performance of the membrane modules was dependent on wastewater composition. Occasionally, non-degradable macromolecular substances concentrated in the bioreactor, resulting in strongly reduced filterability and flow performance of the membrane modules, consequently also reducing the economic viability of the process. The results demonstrate that wastewater-specific pilot tests are absolutely necessary, in particular if the technology is to be used for new applications.

Development and characterization of a small-scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings

A vast majority of pharmacons are beset by possible interactions and side effects which have usually been tested in laboratory animals. However, better methods are needed to reduce the number of animal experiments and interspecies differences with respect to drug metabolism, as well as to provide a faster and more cost-effective way of analysis. These facts have led to the development of in vitro models based on isolated primary hepatocytes to better assess drug metabolism, interactions, and toxicity. A new small-scale bioreactor with the hepatic sandwich model and a gas-permeable membrane at the bottom allowing a definable oxygen exchange, has been constructed and compared with the conventional well plates. Compared to hepatocytes cultured in conventional systems, the cells exhibited a stronger liver-specific capacity and remained in a differentiated state in the small-scale bioreactor over a cultivation period of 17 days. This in vitro model could serve as a tool to predict the liver response to newly developed drugs.

Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.

Evaluation of the optical sulfite oxidation method for the determination of the interfacial mass transfer area in small-scale bioreactors

The optical sulfite oxidation method was recently used by Hermann et al. [R. Hermann, M. Lehmann, J. Büchs, Characterization of gas–liquid mass transfer phenomena in microtiter plates, Biotechnol. Bioeng. 81 (2003) 178–186] and Kensy et al. [F. Kensy, H.F. Zimmermann, I. Knabben, T. Anderlei, H. Trauthwein, U. Dingerdissen, J. Büchs, Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth, Biotechnol. Bioeng. 89 (2005) 698–708.] for determination of interfacial mass transfer area a in a microtiter plate at different shaking intensities. They have determined relative increase of a liquid surface area due to the shaking as a ratio of average specific oxygen absorption rates N ave in 0.5 M sulfite solution with 10 −4 M CoSO 4 under shaken and unshaken conditions. In this paper it is shown that the method is based on the assumption that the profiles of instantaneous oxygen flux Φ during the whole experiments are the same for the shaken and nonshaken solutions. Our experiments performed in a stirred cell did not confirm validity of this assumption. Monitoring of pH and sulfite concentration during the oxidation of sulfite solutions revealed lower values of Φ into nonagitated solution compared to agitated one. The value of N ave in the nonagitated (nonshaken) sulfite solution was by 30% lower than the rate in the agitated one. Consequently, the mass transfer areas measured by the optical sulfite oxidation method are overestimated seriously, cca. 1.4 times. The lower oxygen transfer rate in the nonagitated solution is caused by a local drop of the sulfite concentration near the interface which is due to insufficiently fast transport of sulfite ions from the bulk of a liquid phase to interface under nonagitated (nonshaken) conditions. It is shown that the optical method for measuring of a is fundamentally flawed and should not be used in the presented form.

Confocal Optical System: A Novel Noninvasive Sensor To Study Mixing

A novel confocal optical system to study mixing time in small-scale bioreactors is presented. The system is designed to monitor fluorescence upon tracer addition from a localized confocal volume of 0.21 mL within a glass vessel. The key elements of the fluorescence-based confocal system are a pinhole, a lens, an APD (Avalanche photodiode) detector, and light filters. The optical technique was validated by comparison with a pH-based technique. Finally, the optical sensor was tested and a real cultivation media (i.e., spent mammalian cell media) was used to measure mixing time in a 12.5-mL stirred transparent vessel. High accuracy, easy results interpretation, and low costs are the three most attractive characteristics of the sensor. Because of its noninvasive nature and versatility, the results suggest that the confocal system is a promising tool to perform mixing time studies in stirred vessels.

Low-cost noninvasive optical CO2 sensing system for fermentation and cell culture

High-throughput bioprocessing is a very promising technique for bioprocess development and optimization because of its high efficiency. The key to its development has been the availability of simple and inexpensive sensors to monitor the bioprocesses conducted in its small-scale bioreactors. Here we report on a low-cost noninvasive CO2 sensing system suitable for any transparent vessel. The system was composed of a CO2 sensing patch, a coaster, an interface, and a computer. The sensing film was prepared using the ion-pair technique. The coaster was a small self-made device with necessary optics and electronics for ratiometric measurements with a component cost of less than dollar 100. Results show that the system was stable and reliable despite its simplicity and low cost. The sensitivity of the CO2 sensing system was not affected by pH, media type, or temperature. It was shown to be stable for at least 10 days, long enough for most bioprocesses.

Modeling Composting Rate as a Function of Temperature and Initial Moisture Content

A 5×5×2 factorial design was used to determine the effects of temperature and initial moisture content on the decomposition rate of paper mill sludge with broiler litter composted in a laboratory-scale incubatorbioreactor system. The composting system included ten 4 L volume (filled to 2.76 L) small-scale bioreactors. A first order kinetic model based on CO2 captured using 200 ml of 3.0 N NaOH was used to calculate the decomposition rates. Data on monitored process variables showed that the composting process was not oxygen or moisture limited. Regression analysis applied on the decomposition as a function of temperature and moisture showed that the maximum decomposition occurred around 58°C and 44% w.b. (ash free moisture of 62% w.b.) using the 2-D Gaussian model (R2=0.96). The utilization of this result on operating cost of composting showed that operating the system at 60°C would require 31% of the energy cost of operating at 50°C.

Oxygen uptake rate measurements to monitor the activity of terpene transforming fungi

The application of a simple system for measuring the oxygen uptake rate (OUR) to determine the activity of pellet-forming fungi is described. A non-invasive easy-to-use respirometric system was used to measure the OUR as an activity parameter. The model sensor system was optimized in shake flask experiments with Pleurotus sapidus (DSMZ 8266) and Chaetomium globosum (DSMZ 1962). The toxicity of (+)-limonene and (+)-valencene to submerged fungal cultures and the influence of solvents on C. globosum were investigated. Finally, the new respirometric method was used for the determination of oxygen uptake rates in small scale bioreactors to control the activity of terpene transforming fungi.

Development of a small-scale bioreactor: Application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.

Maximizing cell densities in miniprep-scale cultures with H15 medium and improved oxygen transfer

H15 is a very rich growth medium for culturing bacteria that supports cell densities that are several fold higher than those obtained with more popular media like Luria–Bertani (LB) broth. The performance of H15 is dependent on adequate aeration of the growing culture. This is easily achieved when relatively large baffled flasks are used but not when standard culture tubes are used. As a result, it has been difficult to extend the superior performance of H15 to small-scale bioreactors commonly used by molecular biologists including culture tubes and microtiter plates. Here we describe a simple procedure for improving the oxygen transfer capacity (OTR) of small-scale bioreactors and modifications to the protocol for the preparation of H15 growth medium that make it possible to boost cell density by nearly 400% relative to standard LB broth. Modifications include preparation a glucose-free media stock, sterilization through a 0.45 μm filter rather than autoclaving to avoid the formation of compounds which inhibit cell growth and the introduction of “baffles” for glass culture tubes. Furthermore, these modifications support cell densities in 2 ml that are statistically identical to cell densities obtained in larger baffled flasks. These improvements in the performance and reliability of H15 in small-scale cultures afford researchers the opportunity for significant time and cost savings in the production of plasmid DNA for high-throughput genetic analyses.

Utilization of pretreated molasses for serine alkaline protease production with recombinant bacillus species

Recombinant Bacillus amyloliquefaciens, Bacillus cereus, Bacillus subtilis , and Bacillus licheniformis were used for the production of serine alkaline protease (SAP) utilizing chemically and/or physically pretreated molasses. The highest enzyme activity was obtained with r- Bacillus subtilis , with the complex medium involving physically treated molasses having 20 kg m m 3 initial sucrose concentration in small-scale, agitation- and heating rate-controlled bioreactors at t=63 h. Effects of oxygen transfer were investigated in 3.5 dm 3 laboratory bioreactors under two different agitation rates with r- B. subtilis . Increase in the oxygen transfer rate increased the observed activity and caused the cultivation time of maximum activity shift to the earlier stages of the fermentation. At Q/V=0.5 vvm and N=750 min m 1 , SAP activity reached 2250U cm m 3 at t=36 h.

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Cultivation of Mycobacterium bovis BCG in bioreactors

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.

Optical method for the determination of the oxygen‐transfer capacity of small bioreactors based on sulfite oxidation

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.

Small-scale bioreactor system for process development and optimization

An agitated 12-well microtiter plate system with a working volume of 2 ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO 3-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements. For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader. The setup is simple and inexpensive. The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel.

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor.

A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12-15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100-500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture.