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Fermentation monitoring using in-line and non-invasive spectrometric measurements

Fermentation monitoring using in-line and non-invasive spectrometric measurements. C.A. McGill, A. Nordon and D. Littlejohn Dept of Pure and Applied Chemistry/CPACT, University of Strathclyde, Glasgow, UK. Fermentation process. Streptomyces process

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Fermentation monitoring using in-line and non-invasive spectrometric measurements

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  1. Fermentation monitoring using in-line and non-invasive spectrometric measurements C.A. McGill, A. Nordon and D. Littlejohn Dept of Pure and Applied Chemistry/CPACT, University of Strathclyde, Glasgow, UK

  2. Fermentation process • Streptomyces process • Starting ingredients are carbohydrate, soya protein, oil and trace elements in water • Biomass grown in seed stage then transferred to final stage for production of desired product • Final stage is a fed batch process

  3. Fermentation Process Seed stage - grow biomass Final stage – production of pharmaceutical Transfer biomass Media batching tank

  4. Data currently acquired • On-line (Aspen) • CO2, O2, N2, Ar, pH*, dO2*, RPM*, temp • Off-line • titres*, pH, viscosity • Biochemical analysis • Soluble PO4, CHOs, NH3, proteins, lipids, urea * - final stage only

  5. Aims • Seed stage • optimal transfer time • Final stage • replace off-line measurements • fingerprint batch

  6. Instruments used • Zeiss Corona NIR • Spectraprobe Linx 5-10 MIR

  7. Zeiss Corona NIR

  8. Specification of Zeiss Corona NIR • Reflectance measurements in the 950 – 1700 nm range (6 nm resolution) • Diode array detector • Focal length: 13 mm • 15 detection fibres

  9. SpectraProbe Linx 5-10 MIR Process instrument Lab instrument

  10. Specification of SpectraProbe Linx 5-10 MIR • Probe and spectrometer integrated into a single unit • Hastelloy probe with silicon ATR crystal • 128 element pyro-electric array detector • Chalcogenide fibres transport light to and from sample

  11. Experimental • Seed stage • approx. 50 hours • spectral measurements every 15 min • Final stage • approx. 140 hours • spectral measurements every 15 min

  12. Zeiss Corona monitoring seed stage

  13. SpectraProbe Linx Monitoring the Final Stage

  14. Seed stage • Currently CO2 evolution rate (CER) is used to determine transfer time • This is used for historic reasons – may not be the best time for transfer • Can a spectroscopic technique be used to determine the transfer time?

  15. Seed stage – MIR data Time (possibly C-H bend)

  16. Seed stage – 1st derivative MIR data Time

  17. Seed stage - CER Transfer time

  18. Seed stage – PC1 scores of MIR data Data range: 1400 – 1550 cm-1

  19. Seed stage – MIR results • Small changes in absorbance at ~ 1500 cm-1 • Need to investigate what this change in absorbance means • Can this information be used to determine the optimum time for transferring biomass?

  20. Experimental – final stage • Zeiss Corona NIR • Used NIR data and titre values to construct PLS models for predicting titre • Used NIR data and lipid values to construct PLS models for predicting lipid conc. • Models constructed from data from 6 runs

  21. NIR spectra Time

  22. 1st derivative NIR spectra Time (C-H stretch 2nd overtone) Time (C-H stretch 1st overtone)

  23. Prediction of titre

  24. Prediction of titre

  25. Prediction of Titre Run 1

  26. Prediction of lipids

  27. Prediction of Lipids Run 4

  28. Final Stage - Results • Correlation between NIR data and desired product concentration and lipids concentration • More work required to see if these are direct or indirect correlations • Possible correlations with soluble PO4 and NH3

  29. Overall conclusions • Can possibly use non-invasive NIR instead of off-line analysis to determine titre values or lipids concentration • In-line MIR analysis of the seed stage yields changes in absorbance – needs further investigation

  30. Acknowledgements • GSK Worthing • Barry Barton, Paul Jeffkins, Sarah Stimpson • SpectraProbe Ltd., Clairet Scientific Ltd. • CPACT

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