Abstract
We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.
Similar content being viewed by others
References
FDA (2004) Guidance for Industry, PAT-A Framework for Innovative Pharmaceutical Developement, Manufacturing, and Quality Assurance.
Mazarevica, G., J. Diewok, J. Baena, E. Rosenberg, and B. Lendl (2004) On-line fermentation monitoring by mid-infrared spectroscopy. Appl. Spectrosc. 58: 804–810.
Fernández-Novales, J., M. López, M. Sánchez, J. García, and J. Morales (2008) A feasibility study on the use of a miniature fiber optic NIR spectrometer for the prediction of volumic mass and reducing sugars in white wine fermentations. J. Food Eng. 89: 325–329.
Noguchi, Y., N. Shimba, H. Toyosaki, K. Ebisawa, Y. Kawahara, E. Suzuki, and S. Sugimoto (2002) In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture. J. Microbiol. Methods 51: 73–82.
Noguchi, Y., Y. Nakai, N. Shimba, H. Toyosaki, Y. Kawahara, S. Sugimoto, and E. Suzuki (2004) The energetic conversion competence of Escherichia coli during aerobic respiration studied by 31P NMR using a circulating fermentation system. J. Biochem. 136: 509–515.
Skibsted, E., C. Lindemann, C. Roca, and L. Olsson (2001) Online bioprocess monitoring with a multi-wavelength fluorescence sensor using multivariate calibration. J. Biotechnol. 88: 47–57.
Haack, M., A. Eliasson-Lantz, P. Mortensen, and L. Olsson (2007) Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain. Biotechnol. Bioeng. 96: 904–913.
Ödman, P., C. Lindvald Johansen, L. Olsson, K. V. Gernaey and A. Eliasson Lantz (2009) On-line estimation of biomass, glucose and ethanol in Saccharomyces cerevisiae cultivations using in-situ multi-wavelength fluorescence and software sensors. J. Biotech. 144: 102–112.
Rhiel, M., M. B. Cohen, D. W. Murhammer, and M. A. Arnold (2002) Nondestructive Near-infrared spectroscopic measurement of multiple analytes in undiluted samples of Serum-based cell culture media. Biotechnol. Bioeng. 77: 73–82.
Guilbault, G., B. Danielsson, C. Mandenius, and K. Mosbach (1983) Enzyme electrode and thermistor probes for determination of alcohols with alcohol oxidase. Anal. Chem. 55: 1582–1585.
Alhadeff, E., A. Salgado, O. Cós, N. J. Pereira, F. Valero, and B. Valdman (2008) Integrated biosensor systems for ethanol analysis. Appl. Biochem. Biotechnol. 146: 129–136.
Tarkiainen, V., T. Kotiaho, I. Mattila, I. Virkajärvi, A. Aristidou, and R. Ketola (2005) On-line monitoring of continuous beer fermentation process using automatic membrane inlet mass spectrometric system. Talanta 65: 1254–1263.
Phelps, M., J. Hobbs, D. Kilburn, and R. Turner (1995) An autoclavable glucose biosensor for microbial fermentation monitoring and control. Biotechnol. Bioeng. 46: 514–524.
Esti, M., G. Volpe, D. Compagnone, G. Mariotti, D. Moscone, and G. Palleschi (2003) Monitoring alcoholic fermentation of red wine by electrochemical biosensors. Am. J. Enol. Vitic. 54: 39–45.
Fatt, I. (1976) The polarographic oxygen sensor: Its theory of operation and its application in biology, medicine, and technology. CRC Press, Cleveland.
Diamantis, V., P. Melidis, and A. Aivasidis (2006) Continuous determination of volatile products in anaerobic fermenters by online capillary gas chromatography. Anal. Chim. Acta 573–574: 189–194.
Kokkonen, R., H. Sirén, S. Kauliomäki, S. Rovio, and K. Luomanperä (2004) On-line process monitoring of water-soluble ions in pulp and paper machine waters by capillary electrophoresis. J. Chromatogr A 1032: 243–252.
Sirén, H., K. Luomanperä, T. Työppönen, S. Rovio, P. Vastamäki, and P. Savolahti (2004) Process control and drug analysis with an on-line capillary electrophoresis system. J. Biochem. Biophys. Methods 60: 295–307.
Tahkoniemi, H., K. Helmja, A. Menert, and M. Kaljurand (2006) Fermentation reactor coupled with capillary electrophoresis for on-line bioprocess monitoring. J. Pharm. Biomed. Anal. 41: 1585–1591.
Dinwoodie, R. and D. Mehnert (1985) A continuous method of monitoring and controlling fermentations: Using an automated HPLC system. Biotechnol. Bioeng. 27: 1060–1062.
Turner, C., M. Gregory, and N. Thornhill (1994) Closed-loop control of fed-batch cultures of recombinant Escherichia coli using on-line HPLC. Biotechnol. Bioeng. 44: 819–829.
Koliander, W., C. Arnezeder, and W. Hampel (1990) A simple and versatile system for fermentation control by on-line HPLC analysis of medium components. A Biotechnol. 10: 387–394.
Plum, A. and A. Rehorek (2005) Strategies for continuous online high performance liquid chromatography coupled with diode array detection and electrospray tandem mass spectrometry for process monitoring of sulphonated azo dyes and their intermediates in anaerobic-aerobic bioreactors. J. Chromatogr. A 1084: 119–133.
Rehorek, A. and A. Plum (2006) Online LC-MS-MS process monitoring for optimization of biological treatment of wastewater containing azo dye concentrates. Anal. Bioanal. Chem. 384: 1123–1128.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. F. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, N. van Riel, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk (2000) An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enz. Microb. Technol. 26: 706–714.
Verduyn, C., E. Postma, W. Scheffers, and J. Van Dijken (1992) Effect of benzoic acid on metabolic fluxes in yeast: A continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8: 501–517.
Liu, Y., F. Wang, and W. Lee (2001) On-line monitoring and controlling system for fermentation processes. Biochem. Eng. J. 1: 17–25.
Buttler, T., L. Gorton, H. Jarskog, G. Marko-Varga, B. Hahn-Hägerdal, N. Meinander, and L. Olsson (1994) Monitoring of ethanol during fermentation of a lignocellulose hydrolysate by on-line microdialysis sampling, column liquid chromatography, and an alcohol biosensor. Biotechnol. Bioeng. 44: 322–328.
Rehorek, A., K. Urbig, R. Meurer, C. Schäfer, A. Plum, and G. Braun (2002) Monitoring of azo dye degradation processes in a bioreactor by on-line high-performance liquid chromatography. J. Chromatogr. A 949: 263–268.
Özcan, S. and M. Johnston (1999) Function and regulation of yeast hexose transporters. Microbiol. Mol. Biol. Rev. 63: 554–569.
Reifenberger, E., E. Boles, and M. Ciriacy (1997) Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur. J. Biochem. 245: 324–333.
DeRisi, J. L., V. R. Iyer, and P. O. Brown (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278: 680–686.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to the work.
Rights and permissions
About this article
Cite this article
Tohmola, N., Ahtinen, J., Pitkänen, JP. et al. On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast (Saccharomyces cerevisiae) culture. Biotechnol Bioproc E 16, 264–272 (2011). https://doi.org/10.1007/s12257-010-0147-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-010-0147-3