4.the process

7/26/2019 4.the Process

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Overview

OrganismMedia

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Industrial Microbiology

Handling the process

What is a bioprocessor (fermenter)?


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Outline

Industrial batch cultures

Inoculum development

When do we harvest?

Fed batch cultures

Continuous processes

Characteristics of bioprocessorsAeration and agitation

Ph and temperature control


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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, incrude terms how fast does the process go)

NOTE: Time includes down time, turn-round timeetc.

High Volumetric Productivity minimises thecontribution of fixed coststo the cost of theproduct.


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What are fixed costs?

Fixed costsare business expenses

that are not dependent on the level of

product produced.

They tend to be time-related, such as

salaries Plant, Power, etc.
http://en.wikipedia.org/wiki/Expenseshttp://en.wikipedia.org/wiki/Expenses


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Product formation in a batch

culture

Product

Conc.

Time

Fastestproduction

rate


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How to achieve good

volumetric productivity

Maximise the proportion of time spent at

the fastest production rate by:

Product

Conc.

Time

Fastestproduction

rate


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How to achieve good volumetric

productivity

Minimising the lag before maximum

production starts


Product

Conc.

Time


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productivity

Avoiding subequent phases of slower/zero

production

Choice of harvesting time

Product

Conc.

Time


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productivity

Extending the length of time spent in active

production

Fed batch can do this

Product

Conc.

Time


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productivity

Minimise proportion of time lost as turn-

round time

Fed batch

Continuous processes

Product

Conc.

Time


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How to Achieve Good

Volumetric Productivity

Ensure that production is rapid

Choice of medium and organism

High concentration of active organisms


Product

Conc.

Time

Faster production

= steeper slope


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Key points are:

Inoculum Development

When to Harvest

Extend the Production Phase byFed- Batch

or

Continuous cultures


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Inoculum Development

Inoculum is built up

through a series of stages

Production fermenter is

inoculated with 3-10% ofits total volume

Inoculum contains

A high concentration of

active cells

Ready to commence

maximum production with a

minimal growth requirement


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Advantages of Proper

inoculum Development

High volumetric productivity:

Immediate commencement of productionat maximum rate in the production

fermenter.

A good concentration of active cells

ensures a good production rate..


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Advantages of Proper Inoculum

Development

Balancing growth and production: Optimise inoculum build-up for growthand

production fermenter for production.

Minimise contamination problems.A large healthy inoculum will out-compete

contaminants.

It is economical to discard early stages of build-upwhich are contaminated.

Correct form of fungal mycelium duringproduction. Diffuse or pellets.


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Batch BioprocessesHarvesting

When to harvest for best volumetric productivity

Maximum overall rate of product formation

(remember to include turn-round time)

Product

Conc.

Time

Previous

harvest time


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When to harvest for best titre/yield

First point at which maximum concentration

is reached

Product

Conc.

Time

Previous

harvest time


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NOTEthat the two potential harvesting

points are different

Product

Conc.

Time

Previous

harvest time


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Fed batch culture

Substrates are

pumped into the

fermenter duringthe process

P


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Fed batch culture

Substrates are

pumped into the

fermenter duringthe process

P


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Fed batch culture

What is added?

Medium

Medium componentfor example:

Carbon source

Precursor

When is it added?

To a predetermined programme In response to changes in process variables

pH

O2 concentration


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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 )


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The Crabtree Effect.

1. In the presence of an excess of sugar,

yeasts switch from aerobic to anaerobic

(alcohol producing) metabolism, even underaerobic conditions.

2. High Levels glucose accelerates glycolysis,

produces ethanol rather than biomass bythe TCA cycle


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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 industryCommon

More often used than non-fed cultures?


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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 thesystem divided by the culture volume.

Units of time-1.


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The Chemostat

The system will settle toa steady state, where: Growth rate = dilution

rate (=D)

Growth is nutrient limited Growth is balanced by

loss of cells throughoverflow

Unless the dilution rate

is too high (D>max),when the culture willwash out

Chemostat


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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)


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Overview

OrganismMedia

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Last Thursday: The Process:

Industrial batch cultures

Productivity and Costs


When do we harvest? Fed batch cultures

Started: Continuous processes

Advantages

Disadvantages


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Today:

Recap advantages and disadvantages

of chemostatsChemostats with recycle

Status of Chemostat Culture in Industry

Industrial and Lab-Scale Bioprocessors


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Continuous SystemsIndustrial

Advantages

All the advantages of fed batch

Plus

High volumetric productivity:

In theory,operates continuously at the optimumrate.

In practice, re-establishment (turn round) neededat intervals but less often than batch.

Can handle dilute substrates. Easier to control.

Spreads load on services.


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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.

C ti S t


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Continuous Systems-

Problems

Constant growth means more chance ofmutation/selection.

Chemostats are powerful selection

systems for fitter mutants orcontaminants.

Fitter means able to GROWfaster underculture conditions.

Greater knowledge/familiarity with batchsystems.

C ti S t


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Continuous Systems-

Problems

Existing plant designed for batchoperation.

True continuous operation means

upstream and downstream processingmust also be continuous.

Many (not all) these problems may be

minimised by using cell recycle orretention.

C ti P ith


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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.


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Continuous Processes with Cell

recycle or Retention.

Compared with chemostats, cell

retention or recycle results in:

Higher cell concentrations.

Lower residual substrate concentrations.


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Cell recycle or Retention

Advantages over Chemostats.

Higher volumetric productivity.

Higher cell concentration.

Better yields/titres. Less (or no) substrate needed for growth.

Lower residual substrate concentrations

means less substrate lost through

overflow.

C ll R l R i


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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


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in Industry

Not widespread.

Chemostats only suitable for biomass

production, but valuable in R & D:

Strain selection.

Physiological studies.

Medium optimisation.



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in Industry

Recycle/retention

systems used for:

Biotransformations.

Beverages (withmixed success!).

Effluent treatment:

Continuous supply.

May be dilute. May be poisonous.


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What is a bioprocessor?

A vessel and ancillaries designed to

facilitate the growth and/or activities of

micro-organisms under controlled and

monitored conditions


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Typical Requirements:

Aseptic operation

Agitation and aeration

Measurement and control


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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.


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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

cultures requirements.

The rate of gas to liquid transfer is governedby the gas/liquid interfacial area.

A ti d A it ti i


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Aeration and Agitation in

Conventional Bioprocessors

A sparger bubbles air in at the base of theprocessor 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


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Sizes of BioprocessorNB:Categories etc. are arbitrary!

Working

Volume (L)

Uses

Small 0.5 15 Laboratory,

Experimental

Intermediate 15 1000 Pilot plant,

Experimental,

Production (egtherapeutics)

Large 1000

100,000

Production (bulk

chemicals, antibiotics )


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Production Fermenter

Diagram of

100,000L Fermenter

with:

Top drive agitatorsand foam-breaker


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Diagram of

100,000L Fermenter

with:

Internal cooling coilsand baffles


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Diagram of

100,000L Fermenter

with:

Sparger (air input)

Antibiotic Production


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Fermenters

Installation. Note:

External cooling coils



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Fermenters

Installation. Note:

Location of

mezzanine floor

ANTIBIOTIC PRODUCTION


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FERMENTER

Top (mezzanine

floor). Note:

Agitator motor



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FERMENTER

Top (mezzanine

floor). Note:

Control panel (now

superseded bymicroprocessor/com

puter control)



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FERMENTER

Top (mezzanine

floor). Note:

Inspection hatch



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FERMENTER

Interior view from

bottom. Note:

Agitators



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FERMENTER

Interior view from

bottom. Note:

Baffles



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FERMENTER

Interior view from

bottom. Note:

Inspection hatch and

ladder

ADM CITRIC ACID PLANT


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ADM CITRIC ACID PLANT

(NO-AGITATOR FERMENTERS)

Fermenter BuildingAir mixed fermenters are taller/thinner than

systems with agitators


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CITRIC ACID FERMENTERS

Top

Note lack of agitator motor


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CITRIC ACID FERMENTERS

Base

LARGE PROD FERMENTERS


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LARGE PROD. FERMENTERS

SOME GENERAL POINTS

CIP (clean in place) and in situ

sterilisation

Constructed in stainless steel: Inert and strong

Cooling: Jacket or coils (internal or

external)

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

General View

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Control Consol.

Note:

Microprocessor

logging and control

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Control consol.

Note:

Microprocessor

logging and control Gas supply

rotameters

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Control consol.

Note:

Microprocessor

logging and control Gas supply

rotameters

Pumps for pH

control, antifoam,nutrient feed etc

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Fermenter vessel.

Note:

Detachable stirrer

motor

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Fermenter vessel.

Note:

Detachable stirrer

motor pH/oxygen

electrodes

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Fermenter vessel.

Note:

Detachable stirrer

motor pH/oxygen

electrodes

Exhaust gas

condenser

SMALL AUTOCLAVABLE


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SMALL AUTOCLAVABLE

LAB FERMENTER

Fermenter vessel.Note: Detachable stirrer

motor

pH/oxygenelectrodes

Exhaust gascondenser

Dialysis unit (notusual!)

Lab/research fermenters


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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


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WHAT IS SCALE-UP?

Transferring a process from the lab. (5-20L) 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 facilitatescale-up


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PILOT PLANT FERMENTERS

Usually about one tenth size of productionfermenters and geometrically similar

Half-way house between lab andproduction fermenters

Final optimisation without excessive cost

Supply batches of product for: Downstream processing scale-up

Clinical/field trialsCan also be used for scale down


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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


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Questions (1)

Discuss the use of fed-batch and

continuous bioprocesses in industrial

situations. What are their advantages anddisadvantages when compared

with batch processes?



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a p es o a at o

Questions (2)

What is a fed batch culture and what are

its advantages for the industrial

Microbiologist? Why has its use not beensuperseded by continuous culture?



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Questions (2)

Properties of a useful industrialmicroorganism

(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

4.the process

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