Authors: Vasily Bankovsky, Igor Bankovsky, Pavel Bankovsky

Part IIntroduction

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

RTS-1 — 4 Instruments in 1

Measuring Thermostating

Mixing

SoftwareLogging

Introduction: Noninvasive Mixing Principles

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

Orbital Shaking vs Reverse Spinning

Part IIPrinciples of Reverse Spinning

Growth medium behavior inside the bioreactor

Rotation Intensity Rotation around axis

Spread of the broth media inside of rotation tube as a function of rotation intensity

Vortex behavior inside the tube

Video demonstration:

Reverse Spinning Lifecycle

Centrifugal Forces as a Mechanical Defoamer

Principle of OD measurement

Automatic(A) -

Wavelength of light source: 850nm

Principle of absorption measurement and measurement modes H-L-A

Video demonstration:

Fig. 1 The effect of media volume on layer thickness(mm) during 2000 rpm rotation intensity

Fig. 2

Video demonstration:

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 Results

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. 2 RSL effect on E.coli growth rate over time

Fig. 3 3D representation of Fig. 1

Fig. 4 3D representation of Fig. 1

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. 2 Growth media volume on E.coli growth rate over time

Fig. Growth media volume on E.coli growth kinetics over time in 3D

LB x 2

LB x 1

LB x 1/2

LB x 1/4

LB x 1/8

LB x 4

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)]

Fig. 1 LB conc. effect on E.coli growth kinetics over time

Fig. 2 LB conc. effect on E.coli growth rate over time

Fig. 3 3D representation of Fig. 1

Lactobacillus acidophilus sp. growth kinetics and growth rate [ µ ( 1/h ) vs time (hr)]

• Nitrogen was purged into the bioreactor in sterile conditions to eliminate O2 presence

Fig. 1 LB conc. effect on E.coli growth kinetics over time

Fig. 2 LB conc. effect on E.coli growth rate over time

Fig. 3 3D representation of Fig. 1

Fig. 4 3D representation of Fig. 2

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. 2 T(c) on E.coli growth rate over time

Fig. 3 3D Graph representation of Fig. 1

Fig. 4 3D Graph representation of Fig. 2

Effect of different T-shock (42-70°C) on E.coli BL21 growth kinetics of

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. 3 3D graph of Fig.1

Fig. 4 Temperature in bioreactor over time

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. 3 T-shock effect on E.coli growth rate over time

Fig. 4 3D graph of Fig.1

Fig. 2 Temperature in bioreactor over time

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

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)

Fig. 3 cycling type temperature profiling over time

Possibility to apply different profiling modes: A) Periodic (cycling type) B) Stochastic(random values)C) Mixed type (A+B)

Effect of temperature fluctuation (delta 10°C from 37°C) on growth kinetics and growth rate of E.coli BL21

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

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

Part IVThe Future Developments

Multi-option in 6 Channel Reverse Spinner

RS-4

Color of Phenol Red vs pH after E.coli BL 21 growth on LB and LB+Glucose media

pH= 6 pH= 7 pH=8 LB+Glu LBOptical density vs pH(normalized)

y = 0,976x + 0,0933R2 = 0,9999

0

0,2

0,4

0,6

0,8

1

1,2

0 0,2 0,4 0,6 0,8 1 1,2

570 nm

Linear (570 nm)

pH Indicator Dyes and Micro Color Sensors

Thermophilus sp. cultivation using different non invasive mixing principles

Growth conditions

RS -1 Falcon……...……. ..50 mlTY(0,2%NaCl) ……25 mlSpeed of rotation 2000 rpmReverse ………… 10 secTemperature ….. 60 C

Orbital shaker ES20/60

Falcon …………… 50 mlTY(0,2%NaCl) ……25 mlOrbit ……………….20 mmSpeed of rotation .240 rpmTemperature ……. 60 C

Rocker Falcon……...……. ..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