Small-scale Bioreactor Research Articles

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult. This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 < Re < 2·104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 ≈ 0.3 and P0 ≈ 4.5 for the maximum Reynolds numbers in the different bioreactors.

Studies on Characterization of Mechanically Biologically Treated Waste from Bangalore City

The paper presents results of a comprehensive study on characterization of mechanically biologically treated waste generated in Bangalore, India. Physical, biochemical and engineering characteristics of the waste are presented. The methodology to separate the components of consolidation which includes primary, secondary and biodegradation strains is reviewed. A laboratory scale anaerobic bioreactor depicting the behaviour of the waste in a bioreactor landfill was studied for 370 days and the settlement, gas generation and leachate characteristics were monitored. Study on characterization of waste indicates an organic content of 54% and carbon, nitrogen and hydrogen in the range of 19.28, 1.64 and 11.07% respectively. Around 20–26% of settlement and 15 L of landfill gas/kg of dry waste was observed in laboratory scale bioreactor studies. Both laboratory and small scale bioreactor test results indicate that the MSW has high biodegradable content and biomethane potential. It is prone to large settlements and has shear strength parameters comparable to those published in literature. The behaviour of waste under given conditions can be understood from the above studies and the results provide valuable data necessary for designing MSW treatment scheme which includes waste to energy systems and bio reactor landfill with gas and leachate collection systems.

Product Attribute Forecast: Adaptive Model Selection Using Real-Time Machine Learning

A real-time machine learning framework is developed to forecast product concentration in mammalian cell culture bioreactors. In real-time, the framework evaluates several machine learning algorithms and chooses the most representative algorithm based on current dynamics of the system. Data from multiple sources is combined and only subset of features are fed to the model based on a pre-selection criteria. The model performance is tested using two small-scale bioreactors run. The performance improved towards the end of the process with accumulating data and results for 1 day ahead prediction is presented.

New Small Scale Bioreactor System for the Determination of the Biochemical Methane Potential

Purpose A small scale bioreactor was designed and built to investigate the amount of biogas, and, specifically, methane, produced from different sources.

Sustainable carbon sources for microbial organic acid production with filamentous fungi

BackgroundThe organic acid producer Aspergillus oryzae and Rhizopus delemar are able to convert several alternative carbon sources to malic and fumaric acid. Thus, carbohydrate hydrolysates from lignocellulose separation are likely suitable as substrate for organic acid production with these fungi.ResultsBefore lignocellulose hydrolysate fractions were tested as substrates, experiments with several mono- and disaccharides, possibly present in pretreated biomass, were conducted for their suitability for malic acid production with A. oryzae. This includes levoglucosan, glucose, galactose, mannose, arabinose, xylose, ribose, and cellobiose as well as cheap and easy available sugars, e.g., fructose and maltose. A. oryzae is able to convert every sugar investigated to malate, albeit with different yields. Based on the promising results from the pure sugar conversion experiments, fractions of the organosolv process from beechwood (Fagus sylvatica) and Miscanthus giganteus were further analyzed as carbon source for cultivation and fermentation with A. oryzae for malic acid and R. delemar for fumaric acid production. The highest malic acid concentration of 37.9 ± 2.6 g/L could be reached using beechwood cellulose fraction as carbon source in bioreactor fermentation with A. oryzae and 16.2 ± 0.2 g/L fumaric acid with R. delemar.ConclusionsWe showed in this study that the range of convertible sugars for A. oryzae is even higher than known before. We approved the suitability of fiber/cellulose hydrolysate obtained from the organosolv process as carbon source for A. oryzae in shake flasks as well as in a small-scale bioreactor. The more challenging hemicellulose fraction of F. sylvatica was also positively evaluated for malic acid production with A. oryzae.

Production of dodecanedioic acid via biotransformation of low cost plant-oil derivatives using Candida tropicalis.

Dodecanedioic acid (DDA) is highly useful to the chemical industry as a versatile precursor for producing the polyamide nylon-6,12, which is used for many technical applications, such as heat and chemical-resistant sheaths. However, DDA synthesis has several drawbacks, such as high energy input and cost-intensive removal of by-products. Therefore, alternative bio-based production routes are required due to increasing industrial demand for green chemicals and renewable products. Candida tropicalis converts petrochemical-based n-dodecanes to the corresponding dicarboxylic acids by targeted functionalization. To increase sustainability of the DDA production process, we tested dodecanoic acid methyl ester, which can be easily obtained from transesterification of coconut oil, in whole-cell biotransformation by C. tropicalis. By modifying selected process parameters, a final DDA concentration of 66g/L was achieved using a highly reliable, small-scale bioreactor system. Crucial process development included a gradual pH shift, an optimized substrate feeding strategy, and monitoring the transcriptional profile.

A Perfusion Bioreactor for Making Tissue-Engineered Constructs

Experiments were performed to develop a design for a flow bioreactor. A system was constructed consisting of a small-scale bioreactor placed in a CO2 incubator, with four chambers for making tissue-engineered constructs, providing for long-term experiments in flow conditions maintaining sterility with different humidity and temperature factors in the culture and gaseous media. The adverse influences of flow on cell viability were minimized by optimizing the culture medium flow rate and the excess pressure in the flow chambers. The functional effectiveness of the bioreactor was demonstrated by chondrogenic differentiation of cells in a cell-engineered construct consisting of a microheterogeneous collagen-containing hydrogel matrix biopolymer, mesenchymal stromal cells from human fatty tissue, and chondrogenic differentiation medium as an example.

Comparison of the Biodegradation of n-alkanes and Readily Biodegradable Substrates Using Open Mixed Culture under Aerobic, Anoxic and Anaerobic Conditions

This study has investigated the biodegradation of n-alkanes using open mixed cultures in batch tests. Biodegradation of n-C12, C14, C16, C18, C20 was investigated using a respirometric method and compared with the biodegradation of the readily biodegradable substrates glucose, acetic acid and ethanol. Experiments were performed in small-scale bioreactors under various conditions, i.e. aerobic, anoxic with nitrate and completely anaerobic conditions, using two different sources of open mixed microbial cultures. Under aerobic conditions all the readily biodegradable substrates and hydrocarbons were removed, although the acclimation time was longer for the hydrocarbons (3-5 days) than for the readily biodegradable substrates (1-2 days). No significant effect on the hydrocarbon concentration on biodegradation was observed in the concentration range 0.5-5 g/l. Under anoxic conditions, both the readily biodegradable substrates and the hydrocarbons were removed using nitrate as electron acceptor. However, the acclimation time for the hydrocarbons under anoxic conditions (20-25 days) was much longer than under aerobic conditions. Under both aerobic and anoxic conditions, once acclimation with the hydrocarbons was completed, the microorganisms were immediately able to remove a second spike of the substrate without acclimation. Under anaerobic conditions, no activity was observed with the hydrocarbons over a period of 150 days, while the mixed culture was able to remove glucose and convert it to volatile fatty acids. Under aerobic conditions, the dissolved oxygen consumption data was mathematically modelled using Monod kinetics to obtain biokinetic parameters. Good fittings between the model and the experimental data was obtained and the biodegradation of hydrocarbons was characterised by higher values of the parameter KS compared to the readily biodegradable substrates. The results of this study showed that the open mixed microbial cultures contained diverse microorganisms capable of utilizing both liquid and solid n-alkanes under aerobic and anoxic conditions.

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitin-like polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation.

Quasi-continuous parallel online scattered light, fluorescence and dissolved oxygen tension measurement combined with monitoring of the oxygen transfer rate in each well of a shaken microtiter plate.

BackgroundMicrotiter plates (MTP) are often applied as culture vessels in high-throughput screening programs. If online measuring techniques are available, MTPs can also be applied in the first steps of process development. For such small-scale bioreactors dipping probes are usually too large; therefore, optical measurements are often used. For example, the BioLector technology allows for the online monitoring of scattered light and fluorescence in each well of a continuously orbitally shaken MTP. Although this system provides valuable data, these measurements are mainly of a semi-quantitative nature. Therefore, signal calibration is required to obtain absolute values. With the µRAMOS technology it became possible for the first time to quantify the oxygen transfer rate (OTR) separately in each well of an MTP. In this work, a device is presented that combines both techniques, to provide a hitherto unparalleled high amount of information from each single well.ResultsBecause both systems (BioLector and µRAMOS) are based on optical measurements, the measurements need to be synchronized to avoid interferences with the optical signals. The new experimental setup was applied for online monitoring in cultures of Escherichia coli and Hansenula polymorpha. It has been demonstrated that the well-to-well reproducibility is very high, and that the monitored signals provide reliable and valuable information about the process. With varying filling volumes, different maximum oxygen transfer capacities (OTRmax) were adjusted in oxygen-limited cultures. The different degrees of stress during the culture due to oxygen limitation affected microbial growth and also impacted reproducibility from culture to culture. Furthermore, it was demonstrated that this new device significantly simplifies the experimental efforts: instead of parallel cultures in a shake flask and MTP, just one single experiment in MTP needs to be conducted to measure the OTR, dissolved oxygen tension (DOT), scattered light and fluorescence.ConclusionsThe new device is a very suitable system for the online monitoring of cultures in continuously orbitally shaken MTPs. Due to the high number of parameters that can simultaneously be measured with this small-scale device, deeper insight into the investigated microbial system can be achieved. Furthermore, the experimental efforts to obtain OTR, DOT, scattered light and fluorescence signals during a culture are decreased. Ultimately, this new technology and the resulting high amount of collected data will eliminate the currently existing separation between screening and process development.Graphical abstractPicture of the combined μRAMOS and BioLector setup which allows for measurements of the oxygen transfer rate (OTR), dissolved oxygen tension (DOT), scattered light and fluorescence in each single well of an orbitally shaken microtiter plate. Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0608-2) contains supplementary material, which is available to authorized users.

A flexible well-mixed milliliter-scale reactor with high oxygen transfer rate for microbial cultivations

In order to choose the best strain and subsequently develop an optimal bioprocess many experiments need to be performed. Usually this process is expensive and labor intensive with a limited amount of data available. Small-scale bioreactors and high-throughput platforms are becoming an attractive solution and replacement for existing microtiter plates, shaken flasks and bench scale bioreactors.In this work, a new design of a milliliter-scale bioreactor system is presented and characterized. The entire system consists of a platform with gas connections, heater, temperature sensor and optical fibers on the one side and a bioreactor with special designed magnetic stirrer and non-invasive optical sensors for measurement of pH, dissolved oxygen and optical density on the other side. The system has a high level of flexibility in terms of volume (0.5–2mL), aeration (sparging and surface aeration) and mixing (one- and bi-directional).Computational fluid dynamics (CFD) was employed in order to simulate the mixing times, the oxygen transfer rates and the appearance and size of the gas-liquid interfaces in the 1mL-scale bioreactor with unidirectional mixing and surface aeration.Mixing performance was tested and the oxygen transfer rate was determined experimentally as well. The obtained results show a good mixing time (between 0.4s and 2s) and a high oxygen transfer rate (kLa>1000h−1).The milliliter-scale bioreactor platform was used to cultivate Saccharomyces cerevisiae and Lactobacillus paracasei.

Characterising rhamnolipid production in Burkholderia thailandensis E264, a non-pathogenic producer.

Burkholderia thailandensis E264 is a rhamnolipid (RL)-producing gram-negative bacterium first isolated from the soils and stagnant waters of central and north-eastern Thailand. Growth of B. thailandensis E264 under two different incubation temperatures (25 and 30 °C) resulted in a significantly higher dry cell biomass production at 30 °C (7.71 g/l) than at 25 °C (4.75 g/l) after 264 h; however, incubation at the lower temperature resulted in consistently higher concentration of RL production throughout the growth period. After 264 h, the concentration of crude RL extract for the 25 °C culture was 2.79 g/l compared to 1.99 g/l for the 30 °C culture. Overall RL production concentration after 264 h was 0.258 g/g dry cell biomass (DCB) for the 30 °C culture compared to 0.587 g/g DCB for the 25 °C culture. Real-time PCR (qPCR) was also used to analyse expression of the RL biosynthesis genes throughout the incubation period at 25 °C showing that the expression of the rhlA, rhlB and rhlC genes is continuous. During the log and early stationary phases of growth, expression levels remain low and are increased upon entry to the late stationary phase. B. thailandensis E264 produces mostly di-RLs and the Di-RL C14-C14 in most abundance (41.88 %). Fermentations were also carried out in small-scale bioreactors (4 l working volume) under controlled conditions, and results showed that RL production was maintained. Our findings show that B. thailandensis E264 has excellent potential for industrial scale RL production.

Pharmacological studies of pegylated anti-TNF-α antibodies

Objective Rheumatoid arthritis (RA) is a systemic disease with chronic inflammation of the joints, and is caused by increased inflammatory cytokines such as TNF-α. The inhibition of TNF-α was found to effectively control inflammation, and block the progress of the disease. This study designed a new and efficient strategy to develop TNF-α antibodies. Methods Prokaryotic engineered cells were growing in a small scale bioreactor, and Fab' products (TMP5) were purified by affinity chromatography. Then TMP5 was modified by 20 kDa site-specific pegylation, and purified by SP sepharose column. Activities and affinities of TMP5 and PEG-TMP5 in vitro were studied. Results TMP5 was effectively produced after fermentation, periplasmic extraction and purification. The purity of TMP5 was more than 96%. After pegylation by 20 kDa PEG, the affinity and activity of PEG-TMP5 were (6.48±0.86)×10-9 M and (7.58±0.61)×10-11 M respectively, and were in the same range as positive controls. Conclusion PEG-TMP5 is a promising drug candidate for treating rheumatoid arthritis. Key words: Antibodies; Tumor necrosis factor α; Pegylation; pharmacology in vitro

Small-Scale Perfusion Bioreactor of Red Blood Cells for Dynamic Studies of Cellular Pathways: Proof-of-Concept.

To date, the development of bioreactors for the study of red blood cells (RBCs, daily transfused in the case of disease or hemorrhage) has focused on hematopoietic stem cells. Despite the fact that mature RBCs are enucleated and do not expand, they possess complex cellular and metabolic pathways, as well as post-translation modification signaling and gas-exchange regulation. In order to dynamically study the behavior of RBCs and their signaling pathways under various conditions, a small-scale perfusion bioreactor has been developed. The most advanced design developed here consists of a fluidized bed of 7.6 mL containing 3·109 cells and perfused at 8.5 μL/min. Mimicking RBC storage conditions in transfusion medicine, as a proof-of-concept, we investigated the ex vivo aging of RBCs under both aerobic and anaerobic conditions. Hence, RBCs stored in saline-adenine-glucose-mannitol (SAGM) were injected in parallel into two bioreactors and perfused with a modified SAGM solution over 14 days at room temperature under air or argon. The formation of a fluidized bed enabled easy sampling of the extracellular medium over the storage period used for the quantitation of glucose consumption and lactate production. Hemolysis and microvesiculation increased during aging and were reduced under anaerobic (argon) conditions, which is consistent with previously reported findings. Glucose and lactate levels showed expected trends, i.e., decreased and increased during the 2-week period, respectively; whereas extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolsis and pentose phosphate pathway metabolites, and an accumulation of purine metabolite end-products. This novel approach, which takes advantage of a fluidized bed of cells in comparison to traditional closed bags or tubes, does not require agitation and limit shear stress, and constantly segragates extracellular medium from RBCs. It thus gives access to several difficult-to-obtain on- and off-line parameters in the extracellular medium. This dynamic bioreactor system does not only allow us to probe the behavior of RBCs under different storage conditions, but it also could be a powerful tool to study physiological or pathological RBCs exposed to various conditions and stimuli.

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

Human pluripotent stem cell process parameter optimization in a small scale suspension bioreactor

Background As cell-based therapies begin to enter clinical trials, human pluripotent stem cells (PSCs) present a reliable and robust source of cells for differentiation into all cell types in the human body [1]. PSC may one day provide cells for new treatment options for a wide range of diseases including cardiovascular disease, neurodegenerative diseases, and diabetes. The large numbers of cells required for many of these therapies necessitates the development of scalable and cost efficient technologies for manufacturing PSCs and their derivatives [2]. PSCs are an adherent cell type that is susceptible to phenotypic change in response to environmental stress. Adherent PSCs can be grown in suspension by forming aggregates of undifferentiated cells [3,4], by encapsulating PSCs in beads or hydrogels [5,6], or by adhering PSCs to the surface of microcarriers [7,8]. Most PSC suspension expansion to date has been performed in benchtop stirred-tank bioreactors operating at volumes of 50-200mL. Little bioprocess optimization has been conducted at these scales likely because of the high cost of PSC growth media and labour requirements for bioreactor growth. This study describes the use of a small-scale bioreactor system for Design-of-Experiments based PSC expansion process development. These process developments inform strategies to bring PSC-based processes into clinical production.

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria.

The application of sub-lethal stresses is known to be an efficient strategy to enhance survival of probiotic bacteria during drying processes. In this context, we previously showed that the application of heat stress upon the entry into stationary phase increased significantly the viability of Bifidobacterium bifidum. However, this heat shock has been considered only in small-scale bioreactor and no information is available for a possible scaling-up strategy. Five different operating scales (0.2, 2, 20, 200 and 2000 L) have thus been tested and the results showed that the viability of B. bifidum increases from 3.15 to 6.57 folds, depending on the scale considered. Our observations pointed out the fact that the heat stress procedure is scalable according to the main outcome, i.e., increases in cell viability, but other factors have to be taken into account. Among these factors, dissolved carbon dioxide seems to play a significant role, since it explains the differences observed between the test performed at laboratory scale and in industrial conditions.

Application of multivariate analysis and mass transfer principles for refinement of a 3-L bioreactor scale-down model--when shake flasks mimic 15,000-L bioreactors better.

Characterization of manufacturing processes is key to understanding the effects of process parameters on process performance and product quality. These studies are generally conducted using small-scale model systems. Because of the importance of the results derived from these studies, the small-scale model should be predictive of large scale. Typically, small-scale bioreactors, which are considered superior to shake flasks in simulating large-scale bioreactors, are used as the scale-down models for characterizing mammalian cell culture processes. In this article, we describe a case study where a cell culture unit operation in bioreactors using one-sided pH control and their satellites (small-scale runs conducted using the same post-inoculation cultures and nutrient feeds) in 3-L bioreactors and shake flasks indicated that shake flasks mimicked the large-scale performance better than 3-L bioreactors. We detail here how multivariate analysis was used to make the pertinent assessment and to generate the hypothesis for refining the existing 3-L scale-down model. Relevant statistical techniques such as principal component analysis, partial least square, orthogonal partial least square, and discriminant analysis were used to identify the outliers and to determine the discriminatory variables responsible for performance differences at different scales. The resulting analysis, in combination with mass transfer principles, led to the hypothesis that observed similarities between 15,000-L and shake flask runs, and differences between 15,000-L and 3-L runs, were due to pCO2 and pH values. This hypothesis was confirmed by changing the aeration strategy at 3-L scale. By reducing the initial sparge rate in 3-L bioreactor, process performance and product quality data moved closer to that of large scale.

Progress toward forecasting product quality and quantity of mammalian cell culture processes by performance-based modeling.

The production of biopharmaceuticals requires highly sophisticated, complex cell based processes. Once a process has been developed, acceptable ranges for various control parameters are typically defined based on process characterization studies often comprising several dozens of small scale bioreactor cultivations. A lot of data is generated during these studies and usually only the information needed to define acceptable ranges is processed in more detail. Making use of the wealth of information contained in such data sets, we present here a methodology that uses performance data (such as metabolite profiles) to forecast the product quality and quantity of mammalian cell culture processes based on a toolbox of advanced statistical methods. With this performance based modeling (PBM) the final product concentration and 12 quality attributes (QAs) for two different biopharmaceutical products were predicted in daily intervals throughout the main stage process. The best forecast was achieved for product concentration in a very early phase of the process. Furthermore, some glycan isoforms were predicted with good accuracy several days before the bioreactor was harvested. Overall, PBM clearly demonstrated its capability of early process endpoint prediction by only using commonly available data, even though it was not possible to predict all QAs with the desired accuracy. Knowing the product quality prior to the harvest allows the manufacturer to take counter measures in case the forecasted quality or quantity deviates from what is expected. This would be a big step towards real-time release, an important element of the FDA's PAT initiative.

Automated disposable small-scale bioreactor for high-throughput process development: implementation of the 24 bioreactor array

Aim: A disposable reactor system (250 ml scale) has now been developed into a fully automated 24 reactor array which includes process parameter monitoring and control, advanced feed strategies, automated event triggering, robotic arm liquid handling and sample storage. Results: Process and analytical comparability was shown for a range of Pichia, Escherichia coli and CHO cell-culture bioprocesses up to pilot and commercial scale. This established the system as a scale-down model for process development and characterization of large-scale industrial processes. Conclusion: This work demonstrated the ability of the automated system to accelerate process development by executing a single statistical design of experiments, with a wider range of parameters, up to 3–5-times faster than conventional approaches.