Bioreactor types and their design

Dr. Godfrey Kyazze

University of Westminster

Learning outcomes

• At the end of the lecture you should be able to

– describe the basic functions of a fermenter

– Describe the design of stirred tank reactors, bubble columns and airlift reactors

– Appreciate the challenges posed by culturing of animal cells and how they can be mitigated

– Describe how fermentation parameters can be monitored and controlled and appreciate problems associated with lack of on-line methods for important fermentation parameters.

Generalized schematic of a bioprocess

Substrate + (microbial, plant or animal)

cell or enzyme

process engineering

→→→→→→→→→→→ Products

The heart of a bioprocess used for manufacture of

biologicals is a bioreactor.

Bioreactors (a.k.a fermenters)

Bench top Glass autoclavable

bioreactor

Single use bioreactor

Shaken flask

Production facilities for recombinant protein

Bioreactor

© Fujifilm Diosynth Biotechnologies

(UK) Ltd (bioreactor in background) (Source: Practical Fermentation Technology)

Historical perspective of the use of fermenters

• Alcohol production – Ancient Egypt

• World War I

– Acetone production - C. acetobutylicum

• World War II

– Penicillin production – Penicillium chrysogenum

What is the difference between a fermenter

and a bioreactor?

Production cost in bioprocessing

Fermenter cost in bioprocessing

• For high value products e.g. recombinant proteins and antibodies

– small fraction as most of the money is used for R and D.

– otherwise fermentation costs can make a significant fraction of the processing costs.

Basic functions of a fermenter

Provide a controlled environment for the mass growth ofmicroorganisms or animal cells to obtain a desired product.We need to consider the following:

– sized to provide the required production capacity– Aseptic long term operation– Adequate aeration and agitation but no damage to the organism– Temperature and pH control– able to take samples– suitable for a wide range of processes– minimal use of labour in operation– Enabling factors for GMP e.g. fail-safe systems – power consumption should be as low as possible

Materials of Construction

• Selected to withstand repeated steam sterilization and cleaning cycles

• Non-reactive and non-absorptive

– Examples: glass, stainless steel, plastic (single use bioreactors)

– Glass is non toxic, smooth, corrosion-proof and transparent

Stirred tank reactors

• Most common reactor in bioprocessing

• Main challenge is in provision of oxygen required for growth

• Features– Aspect ratio (simplest height/diameter= 1)

– Impellers (agitators)

– Baffles

– Sparger (types of spargers)

– Heating/ cooling

– Monitoring &Control of foam, temp, pH etc.

(affected by surface area: volume ratio)

• Most common reactor in bioprocessing

• Main challenge is in provision of oxygen required for growth

• Features– Aspect ratio (simplest height/diameter= 1)

– Impellers (agitators)

– Baffles

– Sparger (

– Heating/ cooling

– Monitoring &Control of foam, temp, pH etc.

(affected by surface area: volume ratio)

Stirred tank reactor

Baffles

• reduce vortexing and swirling of liquid in the reactor, improve mixing and reduces “dead spaces”.

Baffles inside a reactor

Stirred tank reactors: Agitation• Agitation objectives:

– O2 transfer

– Nutrient supply

– heating/cooling

– prevent accumulation of toxic metabolites

• Just suspended criterion for suspended solids

• Oxygen transfer affected by type of impeller, its design, mixing speed and size of reactor

Agitation and mixing

• Impellers break the gas into smaller bubbles hence increasing surface area

• What can go wrong?

– Shear

– Flooding

• affects power

consumption

Cavity formation on impellers

Impeller types

Viscosity ranges for different impellers

High viscosity

Low viscosity

Applications of STR

• Applicable where broths are viscous and high oxygen transfer is required

– e.g. manufacture of amino acids, antibiotics, enzymes

Bubble columns

• Aeration and mixing achieved by gas sparging.

• Foaming can be a problem

– Flow pattern affected by

gas flow rate, sparger design, column diameter, viscosity etc.

Bubble column reactor• Applied in production of citric acid and

wastewater treatment

Air-lift reactors

Airlift reactors of different design

• Air is introduced into the

base of the riser by a sparger.

• Driving force for circulation

of medium is the difference in

density in the riser tube (liquid

and many air bubbles) and

the liquid in the down tube

(few air bubbles)

• Applied in production of

bakers yeast, meat substitute

quorn (by the fungus

Fusarium venetatum); growth

of animal cells

Culturing animal cells

• Products of animal cells are very useful

– antibodies, hormones, vaccines

• Animal cells are more nutritionally demanding than microbial cells and are also prone to shear

• doubling times: 12-48 hrs; cell densities <107

cells/ml

• Shear e.g. from impellers or breakup of bubbles on the medium surface

– metabolic changes or cell death

Culturing animal cells• Two ways of culturing animal cells

– anchored onto a support

– submerged in culture

• Anchored cells can be grown in suspension if they are immobilised on microcarriers e.g.

– gelatine

– cellulose

– plastic

Chinese Hamster Ovary (CHO) cells attached

to microcarrier beads

Reactors for animal cells

• Aim to reduce shear; marine propellers at low speed

(10-100rpm) will normally provide adequate mixing

• Round bottomed reactors to ensure better mixing at slow stirrer speeds

• Water jacket for heating to avoid localised heating due to coils.

• Magnetically driven stirrers to reduce risk of contamination

• Encapsulate cells in polymers e.g. calcium alginate

Monitoring and control

• Success of a fermentation depends upon having defined environmental conditions of biomass and product formation.

• need to measure physical and biological activities during the fermentation (monitoring) so that we can take action to give the desired conditions (control).

Need for control

• Reduce variability

• Increase efficiency (by maintaining optimun conditions for product formation)

• Ensure safety

Control terminologyManual and automatic control

Parameters measured or controlled in bioreactors

•Physical Chemical Biological

• Temperature• Pressure• Reactor weight• Liquid level• Foam level• Agitator speed• Power consuption• Gas flow rate• Medium flow rate• Culture viscosity• Gas hold up

• pH• Dissolved oxygen• Dissolved carbon dioxide• Redox potential• Exit gas composition• Conductivity• Broth composition (substrate, product, ion concentrations etc.)

• Biomass concentration• Enzyme concentration• Biomass composition (DNA, RNA, NADH levels etc)• Viability• Morphology

(Source: Doran P. M. 1995. Bioprocess Engineering Principles)

Measurement sensors

• How would you measure the following:

– dissolved oxygen?

– pH?

– biomass?

– Temperature?

• Precautions with measurement instruments

– calibration

– fouling

Dissolved oxygen sensor

pH sensor

Thermocouple

Offline from OD measurements or indirectly from oxygen

consumption rates

Measuring dissolved oxygen concentration

Diffusion of oxygen from the bulk to the cathode

of a dissolved oxygen probe

• Uses dissolved oxygen probes

• galvanic

• polarographic

Polarographic dissolved O2 probe

Control terminology

• Generalised control loop

e = error (set point – value of controlled variable)c = controller outputu = manipulated variable

pH controller from Mettler

Toledo

Control terminology

• Control loop: Temperature control in an endothermic CSTR

In the set up above, which one is the manipulated variable and which one is the final control element?

Controller actions: on/off control

• Final control element is either open or closed

– E.g. thermostat in a house

• Adequate when the demands on the controlled signal are not too strict

• Only effective if they have short time delays

• Not adequate for systems with large sudden changes

PID controllers

• Proportional + Integral + Derivative

• The best control possible – combines all the good bits about P, I and D controllers.

• Widest application.

Summary

• Fementers are designed to mass cultivate cells safely and cost effectively

• All bioreactors must achieve adequate heat transfer, mass transfer, monitoring/control of parameters and are sized to maximize productivity.

• 3 common designs: STR, bubble columns and airlift reactors

• Animal cells are sensitive to shear but can be grown in airlift reactors or on microcarriers.

Review questions

• What factors would you consider in choosing the type of reactor to use for a given fermentation?

• Discuss the importance of monitoring and control of fermentation parameters.

• How would you ensure the following problems do not affect your readings of process variables?

– fouling of pH probes

– foam formation e.g regarding level sensing (what causes foam formation?)

Review questions

• What are the basic functions of a fermenter

• How different are reactors in which aerobic organisms are grown from those in which anaerobic microbes are grown?

• How different is animal cell culture from microbial cell culture?

Further reading

• Stanbury P.F, Whitaker A and Hall S.J. 1995. , Principles of Fermentation Technology. Pergamon, London

• Doran P.M. 1995. Bioprocess Engineering Principles. Academic Press, London.

• McNeil B, Harvey L.M. 2008. Practical Fermentation Technology