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.
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.
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.
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.
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.
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.
Substrates for plasmonic sensors in a flow-through configuration are mostly fabricated by cost-intensive clean room processes, whereas high-volume diagnostic devices are typically made of polymers. This contrast could limit the application of this efficient flow regime in mass-produced devices. In order to become more compatible with polymer processing, a commercially available polycarbonate filter membrane has been evaluated as a substrate for plasmonic flow-through biosensing. The membrane has been sputtered with gold and its sensitivity to changes of bulk refractive index has been determined by transmission measurements using sodium chloride solutions. The sensitivity has been evaluated by determining the wavelength barycenter in a wavelength interval from 470 to 800 nm. The highest determined sensitivity to variations in bulk refractive index is 117 nm RIU −1 (refractive index units). This sensitivity is smaller than that of regular arrays of nanoholes. But the integrating character of the applied evaluation leads to an average standard deviation of 0.005 nm which results in a resolution of 4.1 • 10 −5 RIU. This resolution is sufficient for the detection of protein adsorptions. The proof of principle has been shown with bovine serum albumin and a simplified immunoassay, which consists of the sequential addition of protein A, an IgG antibody and its corresponding antigen. The results show the applicability of this polymeric membrane for biosensing applications. These substrates could enable plasmonic sensing in a flow-through configuration in disposable diagnostic devices.
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