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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky

Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky. Part I Introduction. RTS-1C, Reverse Tube Spinner — Real Time Cell Growth Logger.

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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky

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  1. Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky

  2. Part IIntroduction

  3. RTS-1C, Reverse Tube Spinner — Real Time Cell Growth Logger Specifications: Temperature setting range +4°C ... + 70°C Measurement periodicity till 60 measurements per hour Rotation Speed 250–2000 rpm Software Controlled Experiments 10 units simultaneouslyContinuous Operating Time n/d Warranty 35 000 hrs

  4. RTS-1 — 4 Instruments in 1 Thermostating Measuring Mixing SoftwareLogging

  5. Introduction: Noninvasive Mixing Principles

  6. Reverse Spinning vs Orbital Shaking • Ability to measure OD non invasively • Cell Growth in single use reactors • Natural centric auto-balancing • Simplicity • No power consumption for contra-balancing • Do not need to take aliquot for OD measurement • Proportionality between orbital diameter and the diameter of the moving vessel • Artificial hula-hoop auto-balancing • Larger size • Heavy • Extra power consumption for contra-balancing

  7. Orbital Shaking vs Reverse Spinning

  8. Part IIPrinciples of Reverse Spinning

  9. Growth medium behavior inside the bioreactor Spread of the broth media inside of rotation tube as a function of rotation intensity Rotation around axis Rotation Intensity

  10. Vortex behavior inside the tube Video demonstration:

  11. Reverse Spinning Lifecycle

  12. Centrifugal Forces as a Mechanical Defoamer

  13. Principle of OD measurement Automatic(A) - Wavelength of light source: 850nm

  14. Principle of absorption measurement and measurement modes H-L-A Video demonstration:

  15. Fig. 1 The effect of media volume on layer thickness(mm) during 2000 rpm rotation intensity Video demonstration: Fig. 2

  16. Recommended bioreactor tube: TPP plastic 50mL Tubespin bioreactor • working volume 1 – 30 ml • conical form • 5 openings (A, B, C, D, E) of different size above the gas permeable, sterile PTFE filter of the screw cap • openings can be sealed and by this, exchange adjusted to need • sterile gas exchange is guaranteed by the 0.22 µm filter membrane • even with a high cell density the supply of oxygen through the openings is sufficient • tube fits in a standard 50 ml centrifuge rotor • Single use

  17. Part IIIExperimental Results

  18. Reverse Spin longitude (RSL) effect on E.coli BL21 growth kinetics and growth rate [ µ ( 1/h ) vs time (hr)] Fig. 1 RSL effect on E.coli growth kinetics over time Fig. 3 3D representation of Fig. 1 Fig. 2 RSL effect on E.coli growth rate over time Fig. 4 3D representation of Fig. 1

  19. Growth media volume effect on E.coli BL21 growth kinetics [ 0D(860 nm) vs time (hr)] and growth rate [ µ ( 1/h ) vs time (hr)] Fig. 1 Growth media volume effect on E.coli growth kinetics over time Fig. Growth media volume on E.coli growth kinetics over time in 3D Fig. 2 Growth media volume on E.coli growth rate over time

  20. Influence of LB concentration on E.Coli BL21 growth kinetics [ OD(860 nm) vs time (hr)] and growth rate [ µ ( 1/h ) vs time (hr)] LB x 4 LB x 2 LB x 1 LB x 1/2 LB x 1/4 LB x 1/8 Fig. 3 3D representation of Fig. 1 Fig. 1 LB conc. effect on E.coli growth kinetics over time Fig. 2 LB conc. effect on E.coli growth rate over time

  21. Lactobacillus acidophilus sp. growth kinetics and growth rate [ µ ( 1/h ) vs time (hr)] Fig. 1 LB conc. effect on E.coli growth kinetics over time Fig. 3 3D representation of Fig. 1 Fig. 2 LB conc. effect on E.coli growth rate over time Fig. 4 3D representation of Fig. 2 • Nitrogen was purged into the bioreactor in sterile conditions to eliminate O2 presence

  22. Effect of T(c) on E.coli BL21 growth kinetics [ OD(860 nm) vs time (hr)] and growth rate [µ ( 1/h ) vs time (hr)] Fig. 1 T(c) effect on E.coli growth kinetics over time Fig. 3 3D Graph representation of Fig. 1 Fig. 2 T(c) on E.coli growth rate over time Fig. 4 3D Graph representation of Fig. 2

  23. Effect of different T-shock (42-70°C) on E.coli BL21 growth kinetics of Fig. 3 3D graph of Fig.1 Fig. 1 T-shock effect on E.coli growth kinetics over time Fig. 2 T-shock effect on E.coli growth rate over time Fig. 4 Temperature in bioreactor over time

  24. Effect of T-shock (63…69°C) on E.coli BL21 growth kinetics Fig. 1 T-shock effect on E.coli growth kinetics over time Fig. 2 Temperature in bioreactor over time Fig. 4 3D graph of Fig.1 Fig. 3 T-shock effect on E.coli growth rate over time

  25. Heat treatment optimization by E.coli BL21 growth kinetics investigation of T(C) value pretreatment Fig. 1 3D graph representation of T-shock effect on E.coli growth kinetics over time (42C-70C) Fig. 2 3D graph representation of T-shock effect on E.coli growth kinetics over time (63C-69C) Coarse approximation delta 5 (C) increments from 37(C) to 70(C) Fine approximation with a delta 1(C) increments from 63(C) to 69(C) At 70 (C) irreversible inactivation of 30S subunit of E.coli ribosome take place. ( J.Lee at all.,Appl.Env.Micr. 2002 5379 – 5386 ) Process of inactivation in range 62 (C) till 69 (C) is reversible

  26. Effect of temperature fluctuation (delta 10°C from 37°C) on growth kinetics and growth rate of E.coli BL21 Possibility to apply different profiling modes: A) Periodic (cycling type) B) Stochastic(random values) C) Mixed type (A+B) Fig. 1 t(C) fluctuation effect on E.coli growth kinetics over time Fig. 2 t(C) fluctuation effect on E.coli growth rate over time Fig. 3 cycling type temperature profiling over time

  27. Cultivation of CHO-K1 Cell Culture using Reverse-Spin method

  28. Part IVThe Future Developments

  29. Multi-option in 6 Channel Reverse Spinner

  30. RS-4

  31. Color of Phenol Red vs pH after E.coli BL 21 growth on LB and LB+Glucose media LB+Glu LB pH= 6 pH= 7 pH=8

  32. pH Indicator Dyes and Micro Color Sensors

  33. Thermophilus sp. cultivation using different non invasive mixing principles Growth conditions RS -1 Falcon……...……. ..50 ml TY(0,2%NaCl) ……25 ml Speed of rotation 2000 rpm Reverse ………… 10 sec Temperature ….. 60 C Orbital shaker ES20/60 Falcon …………… 50 ml TY(0,2%NaCl) ……25 ml Orbit ……………….20 mm Speed of rotation .240 rpm Temperature ……. 60 C Rocker Falcon……...……. ..50 ml TY(0,2%NaCl) ……25 ml Angle of rocking ….7 grad Oscillation freq. 120 min-1 Temperature …….60 C Thermophillus sp extremely Difficult to control cells growth in real time because of high optimum of growth temperature ( T(C) is more than 60 C )

  34. New projects with Personal bioreactor RTS-1 • optical pH and Oxygen measurement • Yeast cultivation optimization • Cultivation of microbes associates • Viruses vs cells investigations • Recombinants growth optimization • Environmental models research • Fluorescence detectors for non intrusive analyses of goal fermentation products

  35. Thank you for your attention

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