Andreas Buchenauer

Microfluidic BioLector—Microfluidic Bioprocess Control in Microtiter Plates

In industrial‐scale biotechnological processes, the active control of the pH‐value combined with the controlled feeding of substrate solutions (fed‐batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small‐scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale‐up and scale‐down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small‐scale batches are typically performed highly parallel and in high throughput, large‐scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber‐optic online‐monitoring device for microtiter plates (MTPs)—the BioLector technology—together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed‐batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user‐friendly and can easily be transferred to a disposable single‐use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH‐controlled and fed‐batch conditions in shaken MTPs. Biotechnol. Bioeng. 2010;107: 497–505.

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.

Bioprocess Control in Microscale: Scalable Fermentations in Disposable and User-Friendly Microfluidic Systems

BackgroundThe efficiency of biotechnological production processes depends on selecting the best performing microbial strain and the optimal cultivation conditions. Thus, many experiments have to be conducted, which conflicts with the demand to speed up drug development processes. Consequently, there is a great need for high-throughput devices that allow rapid and reliable bioprocess development. This need is addressed, for example, by the fiber-optic online-monitoring system BioLector which utilizes the wells of shaken microtiter plates (MTPs) as small-scale fermenters. To further improve the application of MTPs as microbioreactors, in this paper, the BioLector technology is combined with microfluidic bioprocess control in MTPs. To realize a user-friendly system for routine laboratory work, disposable microfluidic MTPs are utilized which are actuated by a user-friendly pneumatic hardware.ResultsThis novel microfermentation system was tested in pH-controlled batch as well as in fed-batch fermentations of Escherichia coli. The pH-value in the culture broth could be kept in a narrow dead band of 0.03 around the pH-setpoint, by pneumatically dosing ammonia solution and phosphoric acid to each culture well. Furthermore, fed-batch cultivations with linear and exponential feeding of 500 g/L glucose solution were conducted. Finally, the scale-up potential of the microscale fermentations was evaluated by comparing the obtained results to that of fully controlled fermentations in a 2 L laboratory-scale fermenter (working volume of 1 L). The scale-up was realized by keeping the volumetric mass transfer coefficient kLa constant at a value of 460 1/h. The same growth behavior of the E. coli cultures could be observed on both scales.ConclusionIn microfluidic MTPs, pH-controlled batch as well as fed-batch fermentations were successfully performed. The liquid dosing as well as the biomass growth kinetics of the process-controlled fermentations agreed well both in the microscale and laboratory scale. In conclusion, a user-friendly and disposable microfluidic system could be established which allows scaleable, fully controlled and fully monitored fermentations in working volumes below 1 milliliter.

Surface enhanced infrared spectroscopy with gold strip gratings.

We investigate surface enhanced infrared absorption (SEIRA) spectroscopy with gold strip gratings made by standard optical lithography. By exciting surface plasmon polaritons on both air-gold and gold-substrate interfaces, the resonance of the 1D gratings is linearly tunable with the grating period. With the field enhancement at the edge of the gold strips, a SEIRA enhancement factor more than 6000 for PMMA molecules is achieved. The strong SEIRA enhancement together with the easy fabrication makes the gold strip grating a promising candidate for SEIRA experiments.

Long-term stability of PDMS-based microfluidic systems used for biocatalytic reactions

This paper reports on the changes of PDMS interface characteristics due to the long-term influence of aqueous alkaline solutions, which are frequently used fluids in biocatalytic reactions. Soft lithographic techniques were used to produce polymeric microfluidic systems containing a fluidic layer with multiple cavities for biocatalytic reactions and a pneumatic control layer. The surface energy, the surface roughness and the absorption of liquids on PDMS are analysed as they are important factors affecting the microfluidic current. The results obtained can be used to provide design guidelines for adapting PDMS-based microfluidic devices in long-term bio catalysis reactions.

Microbioreactors with microfluidic control and a user-friendly connection to the actuator hardware

In this study, an array of microbioreactors based on the format of 48-well microtiter plates (MTPs) is presented. The process parameters pH and biomass are monitored online using commercially available optical sensor technology. A microfluidic device dispenses acid or base individually into each well for controlling the pH of fermentations. Fluid volumes from 72 nL to 940 nL can be supplied with valve opening times between 10 ms and 200 ms. One microfluidic device is capable of supplying four wells from two reservoirs. Up to four microfluidic devices can be integrated on the area of a prototype MTP. The devices are fabricated in polydimethylsiloxane (PDMS) using soft lithographic techniques and utilize pneumatically actuated microvalves. During fermentations, the microbioreactor is clamped to an orbital shaker and a temporary pneumatic connection guides the externally controlled pressurized air to the microfluidic device. Finally, fermentations of Escherichia coli in the presence and absence of pH control are carried out in the microbioreactor system over 18 h. During the fermentation the pH of the cultures is continuously monitored by means of optodes. An ammonia solution or phosphoric acid is dispensed to adjust the pH if it differs from the set point of 7.2. In a controlled culture, the pH can be sustained within 7.0 to 7.3 while the pH in an uncontrolled culture ranges between 6.5 and 9.0. This microbioreactor demonstrates the possibility of pH-controlled fermentations in micro-scale. The process control and the user friendly connection to the actuation hardware provide an easy handling comparable to standard MTPs.

Numerical analysis and characterization of bionic valves for microfluidic PDMS-based systems

This paper reports on strong coupled field numerical simulations of flexible artificial micro-machined venous valves. Different designs are optimized towards a maximum pressure distribution onto the flaps of the valves to achieve a higher diodicity. The simulation illustrated mechanisms to elevate the diodicities of the valves. The diodicities of the valves were measured using capacitive-based pressure sensors. The results support the tendencies of the simulated values.

A novel valve for microfluidic PDMS-based systems

This paper reports on an implementation of a novel valve based on the venous valve principle for complex microfluidic networks. Strong coupled field numerical simulations were used to analyse flexible artificial micro-machined venous valves for the first time. Different designs are optimized towards a maximum pressure distribution onto the flaps of the valves to achieve a higher diodicity. The simulation illustrated mechanisms to elevate the diodicities of the valves. The diodicities of the valves were characterised using fluorescence-based measurements, which enables noncontact determination of micro-valve diodicities and volume flows.