Overview Media Organism P R O C E S S Industrial Microbiology Handling the process What is a bioprocessor (fermenter)? Outline  Industrial batch cultures  Inoculum development  When do we harvest?  Fed batch cultures 

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Transcript Overview Media Organism P R O C E S S Industrial Microbiology Handling the process What is a bioprocessor (fermenter)? Outline  Industrial batch cultures  Inoculum development  When do we harvest?  Fed batch cultures 

Overview
Media
Organism
P
R
O
C
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S
Industrial Microbiology
Handling the process
What is a bioprocessor (fermenter)?
Outline
 Industrial
batch cultures
 Inoculum
development
 When do we harvest?
 Fed batch cultures
 Continuous
processes
 Characteristics of bioprocessors
 Aeration
and agitation
 Ph and temperature control
Achieving good volumetric
productivity in a batch system

REMINDER
 Volumetric Productivity

The amount of product produced per unit volume
of production bioprocessor per unit time (or, in
crude terms “how fast does the process go”)

NOTE: “Time” includes down time, turn-round time
etc.

High Volumetric Productivity minimises the
contribution of fixed costs to the cost of the
product.
What are fixed costs?
 Fixed
costs are business expenses
that are not dependent on the level of
product produced.
 They
tend to be time-related, such as
salaries Plant, Power, etc.
Product formation in a batch
culture
Product
Conc.
Fastest
production
rate
Time
How to achieve good
volumetric productivity
Product
Conc.
Fastest
production
rate
Time
 Maximise
the proportion of time spent at
the fastest production rate by:
How to achieve good volumetric
productivity
Product
Conc.
Time
 Minimising
the lag before maximum
production starts

Inoculum development
How to achieve good volumetric
productivity
Product
Conc.
Time
 Avoiding
subequent phases of slower/zero
production

Choice of harvesting time
How to achieve good volumetric
productivity
Product
Conc.
Time
 Extending
the length of time spent in active
production

Fed batch can do this
How to achieve good volumetric
productivity
Product
Conc.
Time
 Minimise
proportion of time lost as turnround time
Fed batch
 Continuous processes

How to Achieve Good
Volumetric Productivity
Product
Conc.
Faster production
= steeper slope
Time
 Ensure
that production is rapid
Choice of medium and organism
 High concentration of active organisms


Inoculum development
Key points are:



Inoculum Development
When to Harvest
Extend the Production Phase by
Fed- Batch
or
Continuous cultures
Inoculum Development



Inoculum is built up
through a series of stages
Production fermenter is
inoculated with 3-10% of
its total volume
Inoculum contains


A high concentration of
active cells
Ready to commence
maximum production with a
minimal growth requirement
Advantages of Proper
inoculum Development
 High
volumetric productivity:
 Immediate
commencement of production
at maximum rate in the production
fermenter.
 A good concentration of active cells
ensures a good production rate..
Advantages of Proper Inoculum
Development

Balancing growth and production:


Minimise contamination problems.



Optimise inoculum build-up for growth and
production fermenter for production.
A large healthy inoculum will out-compete
contaminants.
It is economical to discard early stages of build-up
which are contaminated.
Correct form of fungal mycelium during
production.

Diffuse or pellets.
Batch Bioprocesses –Harvesting
Product
Conc.
Previous
harvest time

Time
When to harvest for best volumetric productivity

Maximum overall rate of product formation
(remember to include turn-round time)
Batch Bioprocesses –Harvesting
Product
Conc.
Previous
harvest time
 When
 First
Time
to harvest for best titre/yield
point at which maximum concentration
is reached
Batch Bioprocesses –Harvesting
Product
Conc.
Previous
harvest time
 NOTE
Time
that the two potential harvesting
points are different
Fed batch culture
P

Substrates are
pumped into the
fermenter during
the process
Fed batch culture
P

Substrates are
pumped into the
fermenter during
the process
Fed batch culture

What is added?
 Medium
 Medium component – for example:



Carbon source
Precursor
When is it added?


To a predetermined programme
In response to changes in process variables


pH
O2 concentration
Fed batch culture

Can be used to extend the production phase
 Substrate may be used as fast as it is added
– concentration in the bioprocess is always
limiting:




Catabolite repression avoided even with readily
used carbon sources (e.g. glucose)
Precursors used efficiently for their correct
purpose
Avoid toxicity problems with some substrates
Efficient yeast biomass production on readily used
carbohydrates (avoiding the Crabtree effect )

1.
2.
The Crabtree Effect.
In the presence of an excess of sugar,
yeasts switch from aerobic to anaerobic
(alcohol producing) metabolism, even under
aerobic conditions.
High Levels glucose accelerates glycolysis,
produces ethanol rather than biomass by
the TCA cycle
Fed batch culture
 Rate
of addition controls rate of use
 Programme
changes in metabolic rate i.e.
can add slow or fast depending on stage of
culture
 Avoid oxygen demand outstripping oxygen
supply
 Status
of fed batch culture in industry
 Common
 More
often used than non-fed cultures?
Continuous Processes

Pump in medium (or substrates).
 Remove culture or spent medium plus
product.
 Types usually encountered in industry:



Simple mixed system with medium input and
culture removal (the Chemostat).
Systems with cell recycle or retention.
Dilution rate (D) is the rate of flow through the
system divided by the culture volume.

Units of time-1.
The Chemostat

The system will settle to
a steady state, where:




Chemostat
Growth rate = dilution
rate (μ =D)
Growth is nutrient limited
Growth is balanced by
loss of cells through
overflow
Unless the dilution rate
is too high (D>μmax),
when the culture will
wash out
The Chemostat



Not used extensively in
industry,
Illustrates the
advantages and
disadvantages of
continuous systems
Disadvantages may be
minimised by the use of
cell recycle or retention
(discussed later)
Overview
Media
Organism
P
R
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Last Thursday: The Process:

Industrial batch cultures





Productivity and Costs
Inoculum development
When do we harvest?
Fed batch cultures
Started: Continuous processes


Advantages
Disadvantages
Today:
 Recap
advantages and disadvantages
of chemostats
 Chemostats with recycle
 Status of Chemostat Culture in Industry
 Industrial and Lab-Scale Bioprocessors
Continuous Systems – Industrial
Advantages

All the advantages of fed batch
Plus
 High volumetric productivity:



In theory,operates continuously at the optimum
rate.
In practice, re-establishment (turn round) needed
at intervals but less often than batch.
Can handle dilute substrates.
 Easier to control.
 Spreads load on services.
Continuous Systems- Problems
 Poor
yields.
 Substrate
constantly needed for growth in
chemostats.
 Unused substrate lost in overflow.
 Generate
large volumes for downstream
processing, often with a poorer titre than
batch systems.
Continuous SystemsProblems
 Constant
growth means more chance of
mutation/selection.
 Chemostats
are powerful selection
systems for “fitter” mutants or
contaminants.
 “Fitter” means able to GROW faster under
culture conditions.
 Greater
knowledge/familiarity with batch
systems.
Continuous SystemsProblems
 Existing
plant designed for batch
operation.
 True continuous operation means
upstream and downstream processing
must also be continuous.
 Many (not all) these problems may be
minimised by using cell recycle or
retention.
Continuous Processes with
Cell recycle or Retention.
 Cells
retained in the bioprocessor or
removed from the effluent and returned.
 Growth rate does not have to equal D
for steady state:
 Growth
rate is less than D.
 Growth rate can, in theory be zero with
100% cell retention.
Continuous Processes with Cell
recycle or Retention.
 Compared
with chemostats, cell
retention or recycle results in:
 Higher
cell concentrations.
 Lower residual substrate concentrations.
Cell recycle or Retention –
Advantages over Chemostats.
 Higher
volumetric productivity.
 Higher
 Better
 Less
cell concentration.
yields/titres.
(or no) substrate needed for growth.
 Lower residual substrate concentrations
means less substrate lost through overflow.
Cell Recycle or Retention –
Advantages over Chemostats.
 Mutation/selection
pressures are less.
 Low
or zero growth.
 Less loss of cells in effluent.
 Less
tendency for culture to wash out.
 Growth
rate does not have to match D.
 Cells are retained.
Status of Continuous Cultures
in Industry
 Not
widespread.
 Chemostats only suitable for biomass
production, but valuable in R & D:
 Strain
selection.
 Physiological studies.
 Medium optimisation.
Status of Continuous Cultures
in Industry

Recycle/retention
systems used for:



Biotransformations.
Beverages (with
mixed success!).
Effluent treatment:



Continuous supply.
May be dilute.
May be poisonous.
What is a bioprocessor?

A vessel and ancillaries designed to
facilitate the growth and/or activities of
micro-organisms under controlled and
monitored conditions
Typical Requirements:
 Aseptic
operation
 Agitation and aeration
 Measurement and control
Aeration and Agitation
 Closely
related (each helps the other).
 Agitation (mixing).
 Provides
uniform, controllable conditions.
 Avoids nutrient depletion and product
build-up around cells.
 Aeration.
 Ensures
oxygen supply to the cells.
Oxygen Supply to Cultures

Cells can only use dissolved oxygen.
 Oxygen is relatively insoluble.
 During a process, oxygen must pass from the
gas phase (air) to the liquid phase (medium)
at a rate which is fast enough to satisfy the
culture’s requirements.
 The rate of gas to liquid transfer is governed
by the gas/liquid interfacial area.
Aeration and Agitation in
Conventional Bioprocessors

A sparger bubbles air in at the base of the
processor



Larger gas/liquid interfacial area
Mixing
Agitators stir the medium


Mixing
Break up bubbles


Larger gas/liquid interfacial area
Increase bubble residence time

Larger gas/liquid interfacial area
Sizes of Bioprocessor
NB: Categories etc. are arbitrary!
Working
Uses
Volume (L)
Small
0.5  15
Laboratory,
Experimental
Intermediate 15  1000 Pilot plant,
Experimental,
Production (eg
therapeutics)
Large
1000 
Production (bulk
100,000
chemicals, antibiotics )
Production Fermenter

Diagram of
100,000L Fermenter
with:

Top drive agitators
and foam-breaker
Production Fermenter

Diagram of
100,000L Fermenter
with:

Internal cooling coils
and baffles
Production Fermenter

Diagram of
100,000L Fermenter
with:

Sparger (air input)
Antibiotic Production
Fermenters

Installation. Note:

External cooling coils
Antibiotic Production
Fermenters

Installation. Note:

Location of
mezzanine floor
ANTIBIOTIC PRODUCTION
FERMENTER

Top (mezzanine
floor). Note:

Agitator motor
ANTIBIOTIC PRODUCTION
FERMENTER

Top (mezzanine
floor). Note:

Control panel (now
superseded by
microprocessor/com
puter control)
ANTIBIOTIC PRODUCTION
FERMENTER

Top (mezzanine
floor). Note:

Inspection hatch
ANTIBIOTIC PRODUCTION
FERMENTER

Interior view from
bottom. Note:

Agitators
ANTIBIOTIC PRODUCTION
FERMENTER

Interior view from
bottom. Note:

Baffles
ANTIBIOTIC PRODUCTION
FERMENTER

Interior view from
bottom. Note:

Inspection hatch and
ladder
ADM CITRIC ACID PLANT
(NO-AGITATOR FERMENTERS)

Fermenter Building

Air mixed fermenters are taller/thinner than
systems with agitators
CITRIC ACID FERMENTERS
 Top
 Note
lack of agitator motor
CITRIC ACID FERMENTERS

Base
LARGE PROD. FERMENTERS –
SOME GENERAL POINTS
 CIP
(clean in place) and in situ
sterilisation
 Constructed in stainless steel:
 Inert
and strong
 Cooling:
external)
Jacket or coils (internal or
SMALL AUTOCLAVABLE LAB
FERMENTER
 General
View
SMALL AUTOCLAVABLE LAB
FERMENTER

Control Consol.
Note:

Microprocessor
logging and control
SMALL AUTOCLAVABLE LAB
FERMENTER

Control consol.
Note:


Microprocessor
logging and control
Gas supply
rotameters
SMALL AUTOCLAVABLE LAB
FERMENTER

Control consol.
Note:



Microprocessor
logging and control
Gas supply
rotameters
Pumps for pH
control, antifoam,
nutrient feed etc
SMALL AUTOCLAVABLE LAB
FERMENTER

Fermenter vessel.
Note:

Detachable stirrer
motor
SMALL AUTOCLAVABLE LAB
FERMENTER

Fermenter vessel.
Note:


Detachable stirrer
motor
pH/oxygen
electrodes
SMALL AUTOCLAVABLE LAB
FERMENTER

Fermenter vessel.
Note:



Detachable stirrer
motor
pH/oxygen
electrodes
Exhaust gas
condenser
SMALL AUTOCLAVABLE LAB
FERMENTER

Fermenter vessel.
Note:




Detachable stirrer
motor
pH/oxygen
electrodes
Exhaust gas
condenser
Dialysis unit (not
usual!)
Lab/research fermenters –
general points
 Monitoring/control
often complex/flexible
 Autoclavable (up to approx 10L)
 Detachable
motor
 Borosilicate glass vessel
 Stainless steel headplate
 In
place sterilisation
 Stainless
steel with sight glass
WHAT IS SCALE-UP?
 Transferring
a process from the lab. (520L) to the factory (possibly 10,000L+)
without loss of optimum characteristics
 Problems include:
Sterility and asepsis
 Inoculum
 Agitation and Aeration

 Pilot
plant may be needed to facilitate
scale-up
PILOT PLANT FERMENTERS
 Usually
about one tenth size of production
fermenters and geometrically similar
 “Half-way house” between lab and
production fermenters
 Final optimisation without excessive cost
 Supply batches of product for:
Downstream processing scale-up
 Clinical/field trials

 Can
also be used for “scale down”
PILOT PLANT FERMENTERS
 Not
always needed:
Low volume/high value added processes
 Computerised optimisation at production level
(no need for scale-down)

Examples of Examination
Questions (1)
Discuss the use of fed-batch and
continuous bioprocesses in industrial
situations. What are their advantages and
disadvantages when compared
with batch processes?
Examples of Examination
Questions (2)
What is a fed batch culture and what are
its advantages for the industrial
Microbiologist? Why has its use not been
superseded by continuous culture?
Examples of Examination
Questions (2)

Properties of a useful industrial
microorganism
 (b) Ethylene oxide sterilization
 (c) Advantages of continuous culture systems
for industrial bioprocesses
 (d) Crude versus defined media for industrial
fermentations
 (e) Depth versus Absolute filters for
sterilisation of air and liquids
 (f) Carbon sources for bioprocesses