In situ sensor techniques in modern bioprocess monitoring

  • Sascha Beutel
  • Steffen Henkel


New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.


In situ sensor Online monitoring Noninvasive measurement 


  1. Anders KD, Wehnert G, Thordsen O, Scheper T, Rehr B, Sahm H (1993) Biotechnological applications of fiber-optic sensing—multiple uses of a fiber-optic fluorometer. Sens Actuators B Chem 11:395–403CrossRefGoogle Scholar
  2. Anton F, Burzlaff A, Kasper C, Brückerhoff T, Scheper T (2007) Preliminary study towards the use of in-situ microscopy for the online analysis of microcarrier cultivations. Eng Life Sci 7:91–96CrossRefGoogle Scholar
  3. Arnold SA, Gaensakoo R, Harvey LM, McNeil B (2002) The use of at-line and in-situ near-infrared spectroscopy to monitor biomass in an industrial fed-batch Escherichia coli process. Biotechnol Bioeng 80:405–413CrossRefGoogle Scholar
  4. Becker T, Hitzmann B, Muffler K, Pörtner R, Reardon KF, Stahl F, Ulber R (2007) Future aspects of bioprocess monitoring. Adv Biochem Eng Biotechnol 150:249–293. doi: 10.1007/10_2006_036 Google Scholar
  5. Bell SEJ, Bourguignon ESO, O’Grady A, Villaumie J, Dennis AC (2002) Extracting Raman spectra from highly fluorescent samples with “Scissors” (SSRS, shifted-substracted Raman spectroscopy). Spectrosc Eur 14:17–20Google Scholar
  6. Bergveld P (1970) Development of an ion-sensitive solid-state device for neurophysiological measurements. IEEE Trans Biomed Eng 19:70–71CrossRefGoogle Scholar
  7. Beutel S, Klein K, Knobbe G, Königfeld P, Petersen K, Ulber R, Scheper T (2002) Controlled enzymatic removal of damaging casein layers on medieval wall-paintings. Biotechnol Bioeng 80:13–21CrossRefGoogle Scholar
  8. Bittner C, Wehnert G, Scheper T (1998) In situ microscopy for on-line determination of biomass. Biotechnol Bioeng 60:24–35CrossRefGoogle Scholar
  9. Bluma A, Höpfner T, Rudolph G, Lindner P, Beutel S, Hitzmann B, Scheper T (2009) Adaptation of in-situ microscopy for crystallization processes. J Cryst Growth 311:4193–4198CrossRefGoogle Scholar
  10. Bluma A, Höpfner T, Lindner P, Rehbock C, Beutel S, Riechers D, Hitzmann B, Scheper T (2010) In-situ imaging sensors for bioprocess monitoring: state of the art. Anal Bional Chem 398:2429–2438CrossRefGoogle Scholar
  11. Bluma A, Höpfner T, Prediger A, Glindkamp A, Beutel S, Scheper T (2011) Process analytical sensors and image-based techniques for single-use bioreactors. Eng Life Sci 11(4):1–4Google Scholar
  12. Bohnke C, Duroy H, Fourquet JL (2003) pH sensors with lithium lanthanum titanate sensitive material: applications in food industry. Sens Actuators B Chem 89:240–247CrossRefGoogle Scholar
  13. Boyd JE, Briskman A, Colvin VL, Mittleman DM (2001) Direct observation of terahertz surface modes in nanometer-sized liquid water pools. Phys Rev Lett 87:14701CrossRefGoogle Scholar
  14. Brecker L, Weber H, Griengl H, Ribbons DW (1999) In situ proton-NMR analyses of Escherichia coli HB101 fermentations in (H2O)-H-1 and in D2O. Microbiol 145:3389–3397Google Scholar
  15. Cannizzaro C, Rhiel M, Marison I, von Stockar U (2003a) On-line monitoring of Phaffia rhodozyma fed-batch process with in situ dispersive Raman spectroscopy. Biotechnol Bioeng 83:668–680CrossRefGoogle Scholar
  16. Cannizzaro C, Gügerli R, Marison I, von Stockar U (2003b) On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy. Biotechnol Bioeng 84:597–610CrossRefGoogle Scholar
  17. Castro CD, Koretsky AP, Domach MM (1999) Performance trade-offs in in situ Chemostat NMR. Biotechnol Prog 15:185–195CrossRefGoogle Scholar
  18. Cervera AE, Petersen N, Lantz AE, Larsen A, Gernaey KV (2009) Application of near-infrared spectroscopy for monitoring and control of cell culture and fermentation. Biotechnol Prog 25:1561–1581Google Scholar
  19. Chen JY, Markelz AG (2003) Towards biosensing with terahertz spectroscopy: ligand binding effects. Biophys J 84:156A–156AGoogle Scholar
  20. Dremel BAA, Schmid RD (1992) Optical sensors for bioprocess control. Chem Ing Tech 64:510–517CrossRefGoogle Scholar
  21. El-Diasty F (2011) Coherent anti-Stokes Raman scattering: spectroscopy and microscopy. Vib Spectrosc 55(1):1–37CrossRefGoogle Scholar
  22. Endres C, Haake C, Landgrebe D, Beutel S, Stahl F, Hitzmann B, Scheper T, Friehs K (2009) Moderne Bioprozessanalytik – eine kurze Übersicht. BIOspektrum 15:662–664Google Scholar
  23. Fernández-Sánchez JF, Cannas R, Spichiger S, Steiger R, Spichiger-Keller UE (2006) Novel nanostructured materials to develop oxygen-sensitive films for optical sensors. Anal Chim Acta 556:271–282CrossRefGoogle Scholar
  24. Ferreira AP, Vieira LM, Cardoso JP, Menezes JC (2005) Evaluation of a new annular capacitance probe for biomass monitoring in industrial pilot-scale fermentations. J Biotechnol 116:403–409CrossRefGoogle Scholar
  25. Gebauer A, Scheper T, Schügerl K (1987) Penicillin acylase production by E. coli. Bioprocess Biosyst Eng 2:55–58Google Scholar
  26. Glindkamp A, Riechers D, Rehbock C, Hitzmann B, Scheper T, Reardon KF (2010) Sensors in disposable bioreactors status and trends. Adv Biochem Eng Biotechnol 115:145–169Google Scholar
  27. Gorry P (1990) General least-squares smoothing and differentiation by the convolution (Savitzki-Golay) method. Anal Chem 62:570–573CrossRefGoogle Scholar
  28. Hagedorn A, Levadoux W, Groleau D, Tartakovsky B (2004) Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production. Biotechnol Prog 20:361–367CrossRefGoogle Scholar
  29. Hantelmann K, Kollecker M, Hüll D, Hitzmann B, Scheper T (2006) Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations. J Biotchnol 121:410–417CrossRefGoogle Scholar
  30. Hatch RT, Veilleux BG (1995) Monitoring of Saccharomyces cerevisiae in commercial bakers' yeast fermentation. Biotechnol Bioeng 46:371–374CrossRefGoogle Scholar
  31. Henning B, Rautenberg J (2006) Process monitoring using ultrasonic sensor systems. Ultrason 44:e1395–e1399CrossRefGoogle Scholar
  32. Herrmann S, Oelßner W, Kaden H, Brischwein M, Wolf B (2000) The influence of different methods of disinfection on the function of electrochemical sensors. Sens Actuators B Chem 69:164–170CrossRefGoogle Scholar
  33. Hiratsuka T (1997) Monitoring the myosin ATPase reaction using a sensitive fluorescent probe: pyrene-labeled ATP. Biophys J 72:843–849CrossRefGoogle Scholar
  34. Joeris K, Frerichs JG, Konstantinov K, Scheper T (2002) In-situ microscopy: online process monitoring of mammalian cell cultures. Cytotech 38:129–134CrossRefGoogle Scholar
  35. Joo S, Brown SB (2008) Chemical sensors with integrated electronics. Chem Rev 108:638–651CrossRefGoogle Scholar
  36. Kadlec P, Gabrys B, Strandt S (2009) Data-driven soft sensors in the process industry. Comput Chem Eng 33:795–814CrossRefGoogle Scholar
  37. Kermis HR, Kostov Y, Rao G (2003) Rapid method for the preparation of a robust optical pH sensor. Analyst 128:1181–1186CrossRefGoogle Scholar
  38. Kitagawa J, Ohkubo T, Onuma M, Kadoya Y (2006) THz spectroscopic characterization of biomolecule/water systems by compact sensor chips. Appl Phys Lett 89:041114CrossRefGoogle Scholar
  39. Klimant I, Kühl M, Glud RN, Holst G (1997) Optical measurement of oxygen and temperature in microscale: strategies and biological applications. Sens Actuators B Chem 38:29–37CrossRefGoogle Scholar
  40. Knüttel T, Meyer H, Scheper T (2006) The application of two-dimensional fluorescence spectroscopy for the on-line evaluation of modified enzymatic enantioselectivities in organic solvents by forming substrate salts. Enzym Microb Technol 39:607–611CrossRefGoogle Scholar
  41. Köneke R, Comte A, Jürgens H, Kohls O, Lam H, Scheper T (1998) Faseroptische Sauerstoffsensoren für Biotechnologie, Umwelt- und Lebensmitteltechnik. Chem Ing Tech 70:1611–1617CrossRefGoogle Scholar
  42. Lai SW, Hou YJ, Che CM, Pang HL, Wong KY, Chang CK, Zhu NY (2004) Electronic spectroscopy, photophysical properties, and emission quenching studies of an oxidatively robust perfluorinated platinum porphyrin. Inorg Chem 43:3724–3732CrossRefGoogle Scholar
  43. Lam H (2002) Entwicklung eines faseroptischen Chemo- und eines Biosensors und deren Einsatz in der Biotechnologie. Dissertation, Universität Hannover, HannoverGoogle Scholar
  44. Landgrebe D, Haake C, Höpfner T, Beutel S, Hitzmann B, Scheper T, Rhiel M, Reardon KF (2010) On-line infrared spectroscopy for bioprocess monitoring. Appl Microbiol Biotechnol 88:11–22CrossRefGoogle Scholar
  45. Lavine B (2000) Chemometrics. Anal Chem 72:91–98CrossRefGoogle Scholar
  46. Leardi B (1994) Application of a genetic algorithm to feature selection under full validation conditions to outliner detection. J Chemometr 8:65–79CrossRefGoogle Scholar
  47. Lee HLT, Boccazzi P, Gorret N, Ram RJ, Sinskey AJ (2004) In situ bioprocess monitoring of Escherichia coli bioreactions using Raman spectroscopy. Vib Spectrosc 35:131–137CrossRefGoogle Scholar
  48. Lee SK, Suh EK, Cho NK, Park HD, Uneus L, Spetz AL (2005) Comparison study of ohmic contacts to 4H-silicon carbide in oxidizing ambient for harsh environment gas sensor applications. Solid State Electron 49:1297–1301CrossRefGoogle Scholar
  49. Lehmann M, Baumann W, Brischwein M, Gahle HJ, Freund I, Ehret R, Drechsler S, Palzer H, Kleintges SU, Wolf B (2001) Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET. Biosens Bioelectron 16:195–203CrossRefGoogle Scholar
  50. Li CY, Zhang XB, Han ZX, Akermark B, Sun LC, Shen GL, Yu RQ (2006) A wide pH range optical sensing system based on a sol-gel encapsulated amino-functionalised corrole. Analyst 131:388–393CrossRefGoogle Scholar
  51. Linder P, Endes C, Bluma A, Höpfner T, Glindkamp A, Haake C, Landgrebe D, Riechers D, Baumfalk R, Hitzmann B, Scheper T, Reardon KF (2011) Disposable sensor systems. In: Eibl R, Eibl D (eds) Single-use technology in biopharmaceutical manufacture. Wiley, New York, pp 67–81CrossRefGoogle Scholar
  52. Marose S, Lindemann C, Ulber R, Scheper T (1999) Optical sensor systems for bioprocess monitoring. Trends Biotechnol 17:30–34CrossRefGoogle Scholar
  53. Matanguihan RM, Konstantinov KB, Yoshida T (1994) Dielectric measurement to monitor the growth and physiological states of biological cells. Bioprocess Biosyst Eng 11:213–222Google Scholar
  54. McGovern AC, Broadhurst D, Taylor J, Kaderbhai N, Winson MK, Small DA, Rowland JJ, Kell DB, Goodacre R (2002) Monitoring of complex industrial bioprocesses for metabolite concentration using modern spectroscopies and machine learning: application to gibberellic acid production. Biotechnol Bioeng 78:527–538CrossRefGoogle Scholar
  55. Miller C (2000) Chemometrics for on-line spectroscopy applications—theory and practice. J Chemom 14:513–528CrossRefGoogle Scholar
  56. Mills A, Chang Q, McMuray N (1992) Equilibrium studies on colorimetric plastic film sensors for carbon dioxide. Anal Chem 64:1383–1388CrossRefGoogle Scholar
  57. Müller JL, Neumann M, Scholl P, Hilterhaus L, Eckstein M, Thum O, Liese A (2010) Online monitoring of biotranformations in high viscous multiphase systems by means of FT-IR and chemometrics. Anal Chem 82:6008–6014CrossRefGoogle Scholar
  58. Munkholm C, Walt DR (1988) A fiber-optic sensor for CO2 measurement. Talanta 35:109–112CrossRefGoogle Scholar
  59. Noui L, Hill J, Keay PJ, Wang RY, Smith T, Yeung K, Habib G, Hoare M (2002) Development of a high resolution UV spectrophotometer for at-line monitoring of bioprocesses. Chem Eng Process 41:107–114CrossRefGoogle Scholar
  60. November EJ, Van Impe JF (2000) Evaluation of on-line viable biomass measurements during fermentation of Candida utilis. Bioprocess Eng 23:473–477CrossRefGoogle Scholar
  61. Ödman P, Lindvald Johansen C, Olsson L, Gernaey KV, Eliasson Lantz A (2010) Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations. Appl Microbiol Biotechnol 86:1745–1759CrossRefGoogle Scholar
  62. Ohkubo T, Onuma J, Kitagawa J, Kadoya Y (2006) Micro-strip-line-based sensing chips for characterization of polar liquids in terahertz regime. Appl Phys Lett 88:212511CrossRefGoogle Scholar
  63. Pekeler T, Lindermann C, Scheper T, Hitzmann B (1998) Prediction of bioprocess from two-dimensional fluorescence specta. Chem Ing Tech 70:1610–1611CrossRefGoogle Scholar
  64. Pons MN, Le Bonte S, Potier O (2004) Spectral analysis and fingerprinting for biomedia characterisation. J Biotechnol 113:211–230CrossRefGoogle Scholar
  65. Potter K, Kleinber RL, Brockmann FJ, McFarland WE (1996) Assay for bacteria in porous media by duffusion weighted NMR. J Magn Reson Ser B 113:9–15CrossRefGoogle Scholar
  66. Prediger A, Bluma A, Höpfner T, Lindner P, Beutel S, Müller JJ, Hilterhaus L, Liese A, Scheper T (2011) In-situ-Mikroskopie zur Online-Überwachung von Enzymträgern und Zweiphasenprozessen. Chem Ing Tech 83:884–887CrossRefGoogle Scholar
  67. Rehbock C, Beutel S, Brückerhoff T, Hitzmann B, Riecher D, Rudolph G, Stahl F, Scheper T, Friehs K (2008) Bioprozessanalytik. Chem Ing Tech 80:267–286CrossRefGoogle Scholar
  68. Resa P, Elvira L, Montero de Espinosa F (2004) Concentration in alcoholic fermentation processes from ultrasonic velocity measurements. Food Res Int 37:587–594CrossRefGoogle Scholar
  69. Rokhina EV, Lens P, Virkutyte J (2009) Low-frequency ultrasound in biotechnology: state of the art. Trends Biotechnol 27:298–306CrossRefGoogle Scholar
  70. Roychoudhury P, Harvey LM, McNeil B (2006) At-line monitoring of ammonium, glucose, methyl oleate and biomass in a complex antibiotic fermentation process using attenuated total reflectance-mid-infrared (ATR-MIR) spectroscopy. Anal Chim Acta 561:218–224CrossRefGoogle Scholar
  71. Rudolph G, Brückerhoff T, Bluma A, Korb G, Scheper T (2007) Optical inline measurement procedure for cell count and cell size determination in bioprocess technology. Chem Ing Tech 79:42–51CrossRefGoogle Scholar
  72. Sato K, Yoshida Y, Hirahara T, Ohba T (2000) On-line measurement of intracellular ATP of Saccharomyces cerevisiae and pyruvate during sake mashing. J Biosci Bioeng 90:294–301Google Scholar
  73. Scheper T, Gebauer A, Sauerbrei A, Niehoff A, Schügerl K (1984) Measurement of biological parameters during fermentation processes. Anal Chim Acta 163:111–118CrossRefGoogle Scholar
  74. Scheper T, Müller C, Anders KD, Eberhardt F, Plötz F, Schelp C, Thordsen O, Schügerl K (1993) Optical sensors for biotechnological applications. Biosens Bioelectron 9:73–83CrossRefGoogle Scholar
  75. Scheper T, Hilmer J, Lammers F, Mueller C, Reinecke M (1996) Biosensors in bioprocess monitoring. J Chromat 725:3–12CrossRefGoogle Scholar
  76. Scheper T, Hitzmann B, Staerk E, Ulber R, Faurie R, Sosnitza P, Reardon KF (1999) Bioanalytics: detailed insight into bioprocesses. Anal Chim Acta 400:121–143CrossRefGoogle Scholar
  77. Schügerl K (2001) Progress in monitoring, modeling and control of bioprocesses during the last 20 years. J Biotechnol 85:149–173CrossRefGoogle Scholar
  78. Schulmann SG, Chen S, Bai F, Leiner MJP, Weis L, Wolfbeis OS (1995) Dependence of fluorescence of immobilized 1-hydroxypyrene-3,6,8-trisulfonate on solution pH: extension of the range of applicability of a ph fluorosensor. Anal Chim Acta 304:165–170CrossRefGoogle Scholar
  79. Severinghaus JW, Bradley AF (1958) Electrodes for blood pO2 and pCO2 determination. J Appl Physiol 13:515–520Google Scholar
  80. Smith AC, Hahn CEW (1975) Studies with Severinghaus pCO2 electrode stability, memory and S plots. Br J Anaesth 47:553–558CrossRefGoogle Scholar
  81. Suhr H, Wehnert G, Schneider K, Bittner C, Scholz T, Geissler P, Jahnr B, Scheper T (1995) In-situ microscopy for online characterization of cell-populations in bioreactors including cell-concentration measurements by depth from focus. Biotechnol Bioeng 47:106–116CrossRefGoogle Scholar
  82. Surribas A, Geissler D, Gierse A, Scheper T, Hitzmann B, Montesinos JL, Valero F (2006) State variables monitoring by in-situ multi-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris. J Biotechnol 124:412–419CrossRefGoogle Scholar
  83. Tan WH, Shi ZY, Kopelman R (1992) Development of submicron chemical fiber optic sensors. Anal Chem 64:2985–2990CrossRefGoogle Scholar
  84. Tartakovsky B, Sheintuch M, Hilmer JM, Scheper T (1996) Application of scanning fluorometry for monitoring of a fermentation process. Biotechnol Prog 12:126–131CrossRefGoogle Scholar
  85. Teixeira AP, Oliveira R, Alves PM, Corrondo MJT (2009) Advances in on-line monitoring and control of mammalian cel cultures: supporting the PAT initiative. Biotechnol Adv 27:726–732CrossRefGoogle Scholar
  86. Trevisan MG, Poppi RJ (2008) Direct determination of ephedrine intermediate in a biotransformation reaction using infrared spectroscopy and PLS. Tatlanta 75:1021–1027CrossRefGoogle Scholar
  87. Ulber R, Fredrichs JG, Beutel S (2003) Optical sensor systems for bioprocess monitoring. Anal Bioanal Chem 376:342–348Google Scholar
  88. Uttamlal M, Walt DR (1995) A fiber-optic carbon dioxide sensor for fermentation monitoring. Biotechnol 13:567–601CrossRefGoogle Scholar
  89. Van Steenkiste F, Baert K, Debruyker D, Spiering V, Van der Schoot B, Arquint P, Born R, Schumann K (1997) A microsensor array for biochemichal sensing. Sens Actuators B Chem 44:409–412CrossRefGoogle Scholar
  90. Vankeirsbilck T, Vercauteren A, Baeyens W, Van der Weken G, Verpoort F, Vergote G, Remon JP (2002) Applications of Raman spectroscopy in pharmaceutical analysis. Trac-Trends Anal Chem 21:869–877CrossRefGoogle Scholar
  91. Voigt H, Schitthelm F, Lange T, Kullick T, Ferretti R (1997) Diamond-like carbon-gate pH-ISFET. Sens Actuators B Chem 44:441–445CrossRefGoogle Scholar
  92. Vojinović V, Cabral JMS, Fonseca LP (2006) Real-time bioprocess monitoring part I: in-situ sensors. Sens Actuators B Chem 114:1083–1091CrossRefGoogle Scholar
  93. Voraberger HS, Kreimeier H, Biebernik K, Kern W (2001) Novel oxygen optrode withstanding autoclavation: technical solutions and performance. Sens Actuators B Chem 74:179–185CrossRefGoogle Scholar
  94. Weigl BH, Wolfbeis OS (1995) Sensitivity studies on optical carbon dioxide sensors based on ion pairing. Sens Actuators B Chem 28:151–156CrossRefGoogle Scholar
  95. Wolfbeis OS (2005) Materials for fluorescence-based optical chemical sensors. J Mater Chem 15:2657–2669CrossRefGoogle Scholar
  96. Yamamoto K, Tominaga K, Sasakawa H, Tamura A, Murakami H, Ohtake H, Sarukura N (2005) Terahertz time-domain spectroscopy of amino acids and polypeptides. Biophys J 89:L22–L24CrossRefGoogle Scholar
  97. Zabriskie DW, Humphrey AE (1978) Estimation of fermentation biomass concentration by measuring culture fluorescence. Appl Environ Microbiol 35:337–343Google Scholar
  98. Zanzotto A, Szita N, Boccazzi P, Lessard P, Sinskey AJ, Jensen KF (2004) Membrane-aerated microbioreactor for high-throughput bioprocessing. Biotechnol Bioeng 87:243–254CrossRefGoogle Scholar
  99. Zeiser A, Bedard C, Voyer R, Jardin B, Tom R, Kamen AA (1999) On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements. Biotechnol Bioeng 63:122–126CrossRefGoogle Scholar
  100. Zhang XC (2002) Terahertz wave imaging: horizons and hurdles. Phys Med Biol 47:3667–3677CrossRefGoogle Scholar
  101. Zhu J, Jin Y, Xue J (1992) Fabrication and characterization of a sterilizable pH-optrode. Fresenius J Anal Chem 342:42–46CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Institute for Technical ChemistryGottfried Wilhelm Leibniz University of HanoverHanoverGermany

Personalised recommendations