Proof-of-concept of a novel micro-bioreactor for fast development of industrial bioprocesses

The experimental performance of a novel micro-bioreactor envisaged for parallel screening and development of industrial bioprocesses has been tested in this work. The micro-bioreactor with an internal volume of 4.5 mL is operated under oscillatory flow mixing (OFM), where a controllable mixing and mass transfer rates are achieved under batch or continuous laminar flow conditions. Several batch fermentations with a flocculent Saccharomyces cerevisiae strain were carried out at initial glucose concentrations (S(0)) range of approximately 5-20 g/L and compared to yeast growth kinetics in a stirred tank (ST) bioreactor. Aerobic fermentations were monitored ex situ in terms of pH, DO, glucose consumption, and biomass and ethanol production (wherever applicable). An average biomass production increase of 83% was obtained in the micro-bioreactor when compared with the ST, with less 93.6% air requirements. It also corresponded to a 214% increase on biomass production when compared with growth in a shaken flask (SF) at S(0) = 20 g/L. Further anaerobic fermentations at the same initial glucose concentration ranges gave the opportunity to use state-of-the-art fiber optics technology for on-line and real-time monitoring of this bioprocess. Time profiles of biomass concentration (measured as optical density (OD)) were very similar in the ST bioreactor and in the micro-bioreactor, with a highly reproducible yeast growth in these two scale-down platforms.     

Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass

Reliable control of the specific growth rate (μ) in fed-batch fermentations depends on the availability of accurate online estimations of the controlled variable. Due to difficulties in measuring biomass, μ is typically estimated using reference models relating measurements of substrate consumption or oxygen uptake rate to biomass growth. However, as culture conditions vary, these models are adapted dynamically, resulting in complex algorithms that lack the necessary robustness for industrial applicability. A simpler approach is presented where biomass is monitored using dielectric spectroscopy. The measurements are subjected to online balances and reconciled in real time against metabolite concentrations and off-gas composition. The reconciled biomass values serve to estimate the growth rate and a simple control scheme is implemented to maintain the desired value of μ. The methodology is developed with the yeast Kluyveromyces marxianus, tested for disturbance rejection and validated with two other strains. It is applicable to other cellular systems with minor modifications.     

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-microm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.     

A new microfluidic concept for parallel operated milliliter-scale stirred tank bioreactors

Parallel miniaturized stirred tank bioreactors are an efficient tool for "high-throughput bioprocess design." As most industrial bioprocesses are pH-controlled and/or are operated in a fed-batch mode, an exact scale-down of these reactions with continuous dosing of fluids into the miniaturized bioreactors is highly desirable. Here, we present the development, characterization, and application of a novel concept for a highly integrated microfluidic device for a bioreaction block with 48 parallel milliliter-scale stirred tank reactors (V = 12 mL). The device consists of an autoclavable fluidic section to dispense up to three liquids individually per reactor. The fluidic section contains 144 membrane pumps, which are magnetically driven by a clamped-on actuator section. The micropumps are designed to dose 1.6 μL per pump lift. Each micropump enables a continuous addition of liquid with a flow rate of up to 3 mL h(-1) . Viscous liquids up to a viscosity of 8.2 mPa s (corresponds to a 60% v/v glycerine solution) can be pumped without changes in the flow rates. Thus, nearly all feeding solutions can be delivered, which are commonly used in bioprocesses. The functionality of the first prototype of this microfluidic device was demonstrated by double-sided pH-controlled cultivations of Saccharomyces cerevisiae based on signals of fluorimetric sensors embedded at the bottom of the bioreactors. Furthermore, fed-batch cultivations with constant and exponential feeding profiles were successfully performed. Thus, the presented novel microfluidic device will be a useful tool for parallel and, thus, efficient optimization of controlled fed-batch bioprocesses in small-scale stirred tank bioreactors. This can help to reduce bioprocess development times drastically.     

BioMEMS: forging new collaborations between biologists and engineers

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons. This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches. This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments. Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.     

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

Introducing fed‐batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed‐batch fermentation processes. Therefore, cost efficient and easy to use small‐scale fed‐batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer‐based controlled‐release fed‐batch microtiter plate is described. In this work, the polymer‐based controlled‐release fed‐batch microtiter plate is used to investigate fed‐batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online‐monitored within each well of the polymer‐based controlled‐release fed‐batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer‐based controlled‐release fed‐batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed‐batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed‐batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale‐up criterion, microtiter plate‐ and shake flask‐based fed‐batch cultivations are highly comparable. On basis of the small‐scale fed‐batch cultivations, a mechanistic model is established and validated. Model‐based simulations coincide well with the experimentally acquired data.     

A new tool for probing of cell–cell communication: human embryonic germ cells inducing apoptosis of SKOV3 ovarian cancer cells on a microfluidic chip

A microfluidic device with unidirectional perfusion has been developed to observe the effect of human embryonic germ (hEG) cells on SKOV3 cells. The hEG and SKOV3 cells were seeded in the inlet and the outlet reservoirs separately, and co-cultured for 2 days. The medium was perfused unidirectionally from the inlet to the outlet. The growth inhibition of SKOV3 cells was monitored online and the apoptosis signals in SKOV3 culture area decreased along the flow of the medium. In conclusion, microfluidic chip is a potentially useful tool to investigate the effect of stem cells on cancer cells with intuitionistic cell-based screens.     

Simple control of fed-batch processes for recombinant protein production with E. coli

A very simple but effective process control technique is proposed that leads to a high batch-to-batch reproducibility with respect to biomass concentration as well as the specific biomass growth rate profiles in E. coli fermentations performed during recombinant protein production. It makes use of the well-established temperature controllers in currently used fermenters, but takes its information from the difference between the controlled culture temperature T (cult) and the temperature T (coolin) of the coolant fed to the fermenter's cooling jacket as adjusted by the fermenter temperature controller. For process control purposes this measured difference is corrected regarding stirrer influences and cumulated before it is used as a new process control variable. As a spin-off of this control, it becomes possible to estimate online the oxygen mass transfer rates and the corresponding k(L)a values during the real cultivation process.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

Assessment of in-line near-infrared spectroscopy for continuous monitoring of fermentation processes

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.     

Evaluation of on-line viable biomass measurements during fermentations of Candida utilis

In this article the suitability of the Biomass Monitor for on-line measurement of viable biomass is thoroughly evaluated during aerobic fermentations of Candida utilis. Successively a number of specifications of the measuring device are discussed for the studied biological system. The optimal measurement frequency for the given experimental conditions is determined. Furthermore, reliable calibrations of the capacitance readings versus well-known off-line analysis of dry weight and plate counts of the yeast have been established. In addition, the impact of varying fermentation conditions such as stirrer speed and air flow rate together with the influence of the oxygen concentration and conductance of the medium on the capacitance signal have been studied and quantified when a significant influence was observed. It is illustrated that knowledge of the viable biomass during fermentations is very useful in the estimation of the specific growth rate of the organism.     

Respiration activity monitoring system for any individual well of a 48-well microtiter plate

Background

Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.

Results

A technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.

Conclusions

The introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.

     

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V_t = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.     

A microfluidic device to establish concentration gradients using reagent density differences

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.     

Evaluation of a new annular capacitance probe for biomass monitoring in industrial pilot-scale fermentations

The four-pin electrode capacitance probe has already shown to be a valuable tool for on-line monitoring viable biomass concentration in industrial-type fermentations. A new prototype annular probe was developed and its performance in real-time monitoring the concentration of viable cells during industrial pilot-scale fermentation for the production of an Active Pharmaceutical Ingredient (API) was investigated and compared to the four-pin probe. A set of 14 fermentations was monitored on-line: four of them with the four-pin probe, the remaining with the annular probe. The performance of both the annular and the four-pin electrode probe were compared against each other and against off-line measurements (viscosity and packed mycelial volume). The prototype annular probe showed to have higher signal intensity and sensitivity than the standard four-pin probe, with higher signal-to-noise ratio. Furthermore, its new design and construction proved to be easier to handle in an industrial environment.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Bacteriocin production with Lactobacillus amylovorus DCE 471 is improved and stabilized by fed-batch fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

Stem cells in microfluidics

With the introduction of microtechnology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and their influence on cell fate studied. Microfluidic devices can be made transparent and the cells monitored real time by imaging, using fluorescence markers to probe cell functions and cell fate. This article gives a perspective on the yet untapped utility of microfluidic devices for stem cell research. It will guide the biologists through some basic microtechnology and the application of microfluidics to cell research, as well as highlight to the engineers the cell culture capabilities of microfluidics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009