Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky

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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel BankovskyPart IIntroductionRTS-1C, Reverse Tube Spinner — Real Time Cell Growth LoggerSpecifications: Temperature…
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Authors: Vasily Bankovsky, Igor Bankovsky, Pavel BankovskyPart IIntroductionRTS-1C, Reverse Tube Spinner — Real Time Cell Growth LoggerSpecifications: 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 hrsRTS-1 — 4 Instruments in 1ThermostatingMeasuringMixingSoftwareLoggingIntroduction: Noninvasive Mixing PrinciplesReverse 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
  • Orbital Shaking vs Reverse SpinningPart IIPrinciples of Reverse SpinningGrowth medium behavior inside the bioreactorSpread of the broth media inside of rotation tube as a function of rotation intensityRotation around axisRotation IntensityVortex behavior inside the tubeVideo demonstration:Reverse Spinning LifecycleCentrifugal Forces as a Mechanical DefoamerPrinciple of OD measurementAutomatic(A) - Wavelength of light source: 850nmPrinciple of absorption measurement and measurement modes H-L-AVideo demonstration:Fig. 1 The effect of media volume on layer thickness(mm) during 2000 rpm rotation intensityVideo demonstration:Fig. 2 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
  • Part IIIExperimental ResultsReverse 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. 1Fig. 2 RSL effect on E.coli growth rate over time Fig. 4 3D representation of Fig. 1Growth 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 3DFig. 2 Growth media volume on E.coli growth rate over time 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 4LB x 2LB x 1LB x 1/2LB x 1/4LB x 1/8Fig. 3 3D representation of Fig. 1Fig. 1 LB conc. effect on E.coli growth kinetics over time Fig. 2 LB conc. effect on E.coli growth rate over time 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. 1Fig. 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
  • 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. 1Fig. 2 T(c) on E.coli growth rate over time Fig. 4 3D Graph representation of Fig. 2Effect of different T-shock (42-70°C) on E.coli BL21 growth kinetics ofFig. 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 Effect of T-shock (63…69°C) on E.coli BL21 growth kineticsFig. 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 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 Effect of temperature fluctuation (delta 10°C from 37°C) on growth kinetics and growth rate of E.coli BL21Possibility 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 timeCultivation of CHO-K1 Cell Culture using Reverse-Spin methodPart IVThe Future DevelopmentsMulti-option in 6 Channel Reverse Spinner RS-4Color of Phenol Red vs pH after E.coli BL 21 growth on LB and LB+Glucose media LB+Glu LBpH= 6 pH= 7 pH=8 pH Indicator Dyes and Micro Color SensorsThermophilus sp. cultivation using different non invasive mixing principles Growth conditionsRS -1Falcon……...……. ..50 mlTY(0,2%NaCl) ……25 mlSpeed of rotation 2000 rpmReverse ………… 10 secTemperature ….. 60 COrbital shaker ES20/60Falcon …………… 50 mlTY(0,2%NaCl) ……25 mlOrbit ……………….20 mmSpeed of rotation .240 rpmTemperature ……. 60 CRockerFalcon……...……. ..50 ml TY(0,2%NaCl) ……25 mlAngle of rocking ….7 gradOscillation freq. 120 min-1Temperature …….60 C Thermophillus sp extremelyDifficult to control cells growth in real time because of high optimum of growth temperature( T(C) is more than 60 C ) 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
  • Thank you for your attention
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