Development of Online Measurement Techniques

 

At the Chair of Bioprocess Engineering, numerous devices have been developed that significantly accelerate bioprocess development on a small culture scale. The RAMOS measurement technology and the development of the BioLector, which have been further developed and commercialised by the companies Hitec Zang, Adolf Kühner AG and m2p-labs GmbH, can be mentioned here exemplarily.

Ramos measurement technology

The RAMOS System (Respiration Activity MOnitoring System) measurement technique allows online monitoring of the respiratory activity of microorganisms (oxygen and carbon dioxide transfer rate and respiration quotient) in up to eight shake flasks as standard^1,2. Parallel operation of up to 16 online monitored shake flasks in one system is also possible. Recording the respiration activity provides the most comprehensive information about a bioprocess and thus accelerates bioprocess development. Conclusions can be drawn about the aerobic metabolic activity of microorganisms and effects, such as oxygen and substrate limitation, product inhibition or diauxia can be detected. The RAMOS measuring technique is continuously being further developed. Thus, in addition to the measurement of the oxygen partial pressure, the measurement of the carbon dioxide partial pressure is also possible. This measurement technique has been successfully implemented in the Kuhner TOM and can be purchased commercially in the future. The AnaRAMOS and SynRAMOS can be used to monitor the growth of anaerobic microorganisms, as well as microorganisms growing on gaseous carbon sources^3,4. Monitoring of exotic gases such as ethylene is also possible⁵. The RAMOS measurement technique was originally developed for shake flasks and could be successfully transferred to the microplate scale in the form of µRAMOS⁶.

COSBIOS

With the COSBIOS system (Continuously Operated Shaken BIOreactor System), biological process development and characterization of a continuously operated process in shake flask-scale can be performed^7,8. The obtained fermentation data on the product formation characteristics of the cultivated microorganisms are extremely valuable for the design of a process in continuous mode. The system consists of eight glass cylinders, each with an overflow. The system is gassed through the headspace via two separate ports and supplied with a feed solution. The filling volume is only 15-25 mL. After reaching a steady state, eight values of a so-called X/D-diagram are already available. Compared to individual stirred tank reactors, which have to be operated successively at different flow rates, the operation of the COSBIOS system is more time-, cost- and resource-efficient.

ViMOS

In some microbial culture systems, a change in viscosity can be observed over the course of the cultivation. The cause may be the dissolution of polymeric media components or the formation of biopolymers. Especially in the biological process development of filamentous growing microorganisms, the change of viscosity over the time course of the cultivation is a central aspect. The ViMOS system (Viscosity MOnitoring System) makes it possible to measure the viscosity in eight shake flasks online and contact-free⁹. Here, the phenomenon is used that as the viscosity of the cultivation broth increases, there is an increasing relative phase shift between the movement of the leading edge of the bulk liquid in the shake flask and the circular direction of the centrifugal acceleration. The phase angle of the liquid is detected here via an optical setup. This makes it possible, based on a previously performed calibration, to calculate back to the viscosity. The ViMOS technique can easily be coupled with the RAMOS technique.

BioLector

The BioLector technique, which allows online spectroscopic monitoring of shaken fermentations in microplates, was invented at the Department of Biochemical Engineering¹⁰. The system consists of a cultivation chamber with a shaker, on which a microtiter plate is placed, and a spectrometer with an optical fiber, which is attached to a X-Y positioning device. The positioning device moves the optical fiber under the individual wells of the microplate one after the other during operation, so that the wells can be measured spectroscopically. From the data of the scattered light and fluorescence recorded in this way, the biomass concentration and biogenic fluorescence of e.g. tryptophan, NADH or riboflavin can be characterized online. If expressed target proteins are fused (tagged) with fluorescent proteins, product formation can be monitored online. Commercially available sensor spots can also be used to monitor oxygen saturation and pH in the fermentation broth online. At the AVT.BioVT, the BioLector technology was coupled with a pipetting robot for the first time (RoboLector)¹¹. This allows fully automated experiments^12,13,14,15. Furthermore, the basic principle of the so-called BioLectorPro technique was developed with a cooperation partner. Here, microplates are used, which are equipped with a special microfluidic system^16,17. In this way, pH-regulated cultivations or fed-batch cultivations with a freely selectable feeding profile can be realized. For cultivations without oxygen limitation in the BioLector, the so-called flowerplate was developed¹⁸. Different baffled well geometries were optimized according to various criteria. The flowerplate offers maximum oxygen transfer capacities close to those of stirred reactors. With the help of a custom-built temperature block, a temperature gradient can also be applied across a microplate. Thus, the temperature dependence of microbiological systems can be studied in one single experiment¹⁹.

A combination of the BioLector with the µRAMOS technique has also resulted in a device that additionally allows the monitoring of the respiratory activity of the microorganisms²⁰.Another noteworthy development of the BioLector is the 2D-BioLector. This device is capable to measure not only defined individual wavelength combinations, but also entire 2D spectra in all wells over time²¹. This provides an extremely large amount of process information. This allows to predict the concentrations of non-optically reacting substances such as glucose or glycerol.

Monitoring of fermentation processes

In addition to the development of entire measurement systems, individual components and new approaches for monitoring and characterizing fermentation processes are also being developed at the AVT.BioVT. The following list gives examples of some developments in this field:

• Measurement of dissolved-CO concentration in syngas fermentation in stirred-tank reactors²².
• Characterization of microaerophilic processes²³.
• Online determination of viscosity in 50 L pressure fermenter via calorimetric measurements²⁴.

If you have any questions, feel free to contact us via the indicated contact person.

We continuously offer theses on the mentioned topics. You can find student and final theses here (access only from the RWTH network). In addition, you are welcome to send us a spontaneous application via our secretary.

 

References

¹ Anderlei, T., & Büchs, J. (2001). Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochemical Engineering Journal, 7(2), 157-162.

² Anderlei, T., Zang, W., Papaspyrou, M., & Büchs, J. (2004). Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochemical Engineering Journal, 17(3), 187-194.

³ Mann, M, Wittke, D, Büchs, J. Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for Clostridium ljungdahlii. Eng Life Sci. 2020; 1‐ 10. https://doi.org/10.1002/elsc.202000054

⁴ Mann, M., Hüser, A., Schick, B., Dinger, R., Miebach, K., & Büchs, J. (2021). Online monitoring of gas transfer rates during CO and CO/H₂ gas fermentation in quasi‐continuously ventilated shake flasks. Biotechnology and Bioengineering, 1– 13. https://doi.org/10.1002/bit.27722

⁵ Schulte, A., Schilling, J.V., Nolten, J. et al. Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device. BMC Plant Biol 18, 101 (2018). https://doi.org/10.1186/s12870-018-1305-6

⁶ Flitsch, D., Krabbe, S., Ladner, T., Beckers, M., Schilling, J., Mahr, S., ... & Büchs, J. (2016). Respiration activity monitoring system for any individual well of a 48-well microtiter plate. Journal of Biological Engineering, 10(1), 1-14.

⁷ Sieben, M., Steinhorn, G., Müller, C., Fuchs, S., Ann Chin, L., Regestein, L., & Büchs, J. (2016). Testing plasmid stability of Escherichia coli using the continuously operated shaken BIOreactor system. Biotechnology Progress, 32(6), 1418-1425.

⁸ Akgün, A., Müller, C., Engmann, R. et al. Application of an improved continuous parallel shaken bioreactor system for three microbial model systems. Bioprocess Biosyst Eng 31, 193–205 (2008). https://doi.org/10.1007/s00449-007-0183-3

⁹ Sieben, M., Hanke, R., & Büchs, J. (2019). Contact-free determination of viscosity in multiple parallel samples. Scientific reports, 9(1), 1-10.

¹⁰ Samorski, M., Müller‐Newen, G., & Büchs, J. (2005). Quasi‐continuous combined scattered light and fluorescence measurements: A novel measurement technique for shaken microtiter plates. Biotechnology and Bioengineering, 92(1), 61-68.

¹¹ Huber, R., Ritter, D., Hering, T. et al. Robo-Lector – a novel platform for automated high-throughput cultivations in microtiter plates with high information content. Microb Cell Fact 8, 42 (2009). https://doi.org/10.1186/1475-2859-8-42

¹² Mühlmann, M., Kunze, M., Ribeiro, J. et al. Cellulolytic RoboLector – towards an automated high-throughput screening platform for recombinant cellulase expression. J Biol Eng 11, 1 (2017). https://doi.org/10.1186/s13036-016-0043-2

¹³ Mühlmann, M., Forsten, E., Noack, S. et al. Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Fact 16, 220 (2017). https://doi.org/10.1186/s12934-017-0832-4

¹⁴ Mühlmann, M.J., Forsten, E., Noack, S. and Büchs, J. (2018), Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden. Biotechnol. Prog., 34: 1543-1552. https://doi.org/10.1002/btpr.2704

¹⁵ Mühlmann, M., Büchs, J. Automatisiertes Klonscreening und Vorhersage der Expressionsleistung. Biospektrum 24, 46–49 (2018). https://doi.org/10.1007/s12268-018-0891-z

¹⁶ Funke, M., Buchenauer, A., Schnakenberg, U., Mokwa, W., Diederichs, S., Mertens, A., Müller, C., Kensy, F. and Büchs, J. (2010), Microfluidic biolector—microfluidic bioprocess control in microtiter plates. Biotechnol. Bioeng., 107: 497-505. https://doi.org/10.1002/bit.22825

¹⁷ Funke, M., Buchenauer, A., Mokwa, W. et al. Bioprocess Control in Microscale: Scalable Fermentations in Disposable and User-Friendly Microfluidic Systems. Microb Cell Fact 9, 86 (2010). https://doi.org/10.1186/1475-2859-9-86

¹⁸ Funke, M., Diederichs, S., Kensy, F., Müller, C. and Büchs, J. (2009), The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations. Biotechnol. Bioeng., 103: 1118-1128. https://doi.org/10.1002/bit.22341

¹⁹ Kunze, M., Lattermann, C., Diederichs, S. et al. Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes. J Biol Eng 8, 22 (2014). https://doi.org/10.1186/1754-1611-8-22

²⁰ Ladner, T., Held, M., Flitsch, D., Beckers, M., & Büchs, J. (2016). 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. Microbial Cell Factories, 15(1), 206.

²¹ Ladner, T., Beckers, M., Hitzmann, B., & Büchs, J. (2016). Parallel online multi‐wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate. Biotechnology Journal, 11(12), 1605-1616.

²² Mann, M, Miebach, K, Büchs, J. Online measurement of dissolved carbon monoxide concentrations reveals critical operating conditions in gas fermentation experiments. Biotechnology and Bioengineering. 2020; 1– 12. https://doi.org/10.1002/bit.27567

²³ Heyman, B., Tulke, H., Putri, S. P., Fukusaki, E., & Büchs, J. (2020). Online monitoring of the respiratory quotient reveals metabolic phases during microaerobic 2, 3‐butanediol production with Bacillus licheniformis. Engineering in Life Sciences, 20(3-4), 133-144.

²⁴ Schelden, M., Lima, W., Doerr, E. W., Wunderlich, M., Rehmann, L., Büchs, J., & Regestein, L. (2017). Online measurement of viscosity for biological systems in stirred tank bioreactors. Biotechnology and Bioengineering, 114(5), 990-997.