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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

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
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
fermentation process3
Fermentation Process

Seed stage - grow biomass

Final stage – production of pharmaceutical

Transfer biomass

Media batching tank

data currently acquired
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

slide5
Aims
  • Seed stage
    • optimal transfer time
  • Final stage
    • replace off-line measurements
    • fingerprint batch
instruments used
Instruments used
  • Zeiss Corona NIR
  • Spectraprobe Linx 5-10 MIR
specification of zeiss corona nir
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
spectraprobe linx 5 10 mir
SpectraProbe Linx 5-10 MIR

Process instrument

Lab instrument

specification of spectraprobe linx 5 10 mir
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
experimental
Experimental
  • Seed stage
    • approx. 50 hours
    • spectral measurements every 15 min
  • Final stage
    • approx. 140 hours
    • spectral measurements every 15 min
seed stage
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?
seed stage mir data
Seed stage – MIR data

Time

(possibly C-H bend)

seed stage cer
Seed stage - CER

Transfer time

seed stage pc1 scores of mir data
Seed stage – PC1 scores of MIR data

Data range: 1400 – 1550 cm-1

seed stage mir results
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?
experimental final stage
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
1 st derivative nir spectra
1st derivative NIR spectra

Time

(C-H stretch

2nd overtone)

Time

(C-H stretch

1st overtone)

final stage results
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
overall conclusions
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
acknowledgements
Acknowledgements
  • GSK Worthing
    • Barry Barton, Paul Jeffkins, Sarah Stimpson
  • SpectraProbe Ltd., Clairet Scientific Ltd.
  • CPACT