Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes.

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high-throughput screening and optimization of process conditions. In addition, advances in the bioprocessing industry have led to increasingly complex process designs, such as pH and temperature shifts, in microbial fed-batch fermentations for optimal soluble protein expression in a range of hosts. However, in order to develop an accurate scale-down model for bioprocess screening and optimization, small-scale bioreactors must be able to accurately reproduce these complex process designs. Monitoring methods, such as fluorometric-based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Subsequently, we present our calibration methodology for more precise online measurement and provide recommendations for improved pH control in sophisticated fermentation processes.

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening.

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed-batch processes, including at-line sampling, deep freezer off-line sample storage, and complete data handling. To enable scalable high-throughput early-stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom-built hardware and software, fully automated milliliter-scale microbial fed-batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high-throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early-stage bioprocess development.

Evaluation of fluorimetric pH sensors for bioprocess monitoring at low pH.

Optical chemical sensors are the standard for pH monitoring in small-scale bioreactors such as microtiter plates, shaking flasks or other single-use bioreactors. The dynamic pH range of the so far commercially available fluorescent pH sensors applied in small-scale bioreactors is restricted to pH monitoring around neutral pH, although many fermentation processes are performed at pH < 6 on industrial scale. Thus, two new prototype acidic fluorescence pH sensors immobilized in single-use stirred-tank bioreactors, one with excitation at 470 nm and emission at 550 nm (sensor 470/550) and the other with excitation at 505 nm and emission at 600 nm (sensor 505/600), were characterized with respect to dynamic ranges and operational stability in representative fermentation media. Best resolution and dynamic range was observed with pH sensor 505/600 in mineral medium (dynamic range of 3.9 < pH < 7.2). Applying the same pH sensors to complex medium results in a drastic reduction of resolution and dynamic ranges. Yeast extract in complex medium was found to cause background fluorescence at the sensors' operating wavelength combinations. Optical isolation of the sensor by adding a black colored polymer layer above the sensor spot and fixing an aperture made of adhesive photoresistant foil between the fluorescence reader and the transparent bottom of the polystyrene reactors enabled full re-establishment of the sensor's characteristics. Reliability and operational stability of sensor 505/600 was shown by online pH monitoring (4.5 < pH < 5.8) of parallel anaerobic batch fermentations of Clostridium acetobutylicum for the production of acetone, butanol and ethanol (ABE) with offline pH measurements with a standard glass electrode as reference.

Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors.

Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.