Overview Organism Media PROCESSPROCESS

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.expenses  They tend to be time-related, such as salaries Plant, Power, etc.

Product formation in a batch culture Product Conc. Time Fastest production rate

How to achieve good volumetric productivity  Maximise the proportion of time spent at the fastest production rate by: Product Conc. Time Fastest production rate

How to achieve good volumetric productivity  Minimising the lag before maximum production starts  Inoculum development Product Conc. Time

How to achieve good volumetric productivity  Avoiding subequent phases of slower/zero production  Choice of harvesting time Product Conc. Time

How to achieve good volumetric productivity  Extending the length of time spent in active production  Fed batch can do this Product Conc. Time

How to achieve good volumetric productivity  Minimise proportion of time lost as turn- round time  Fed batch  Continuous processes Product Conc. Time

How to Achieve Good Volumetric Productivity  Ensure that production is rapid  Choice of medium and organism  High concentration of active organisms Inoculum development Product Conc. Time Faster production = steeper slope

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:  Optimise inoculum build-up for growth and production fermenter for production.  Minimise contamination problems.  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  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

Batch Bioprocesses –Harvesting  When to harvest for best titre/yield  First point at which maximum concentration is reached Product Conc. Time Previous harvest time

Batch Bioprocesses –Harvesting  NOTE that the two potential harvesting points are different Product Conc. Time Previous harvest time

Fed batch culture  Substrates are pumped into the fermenter during the process P

Fed batch culture  Substrates are pumped into the fermenter during the process P

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  O 2 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 )

 The Crabtree Effect. 1. In the presence of an excess of sugar, yeasts switch from aerobic to anaerobic (alcohol producing) metabolism, even under aerobic conditions. 2. 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:  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>μ max ), when the culture will wash out Chemostat

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 Organism Media PROCESSPROCESS

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 Systems- Problems  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 Systems- Problems  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 cell concentration.  Better yields/titres.  Less (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 Volume (L) Uses Small0.5  15Laboratory, Experimental Intermediate15  1000Pilot plant, Experimental, Production (eg therapeutics) Large1000  100,000 Production (bulk 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: Jacket or coils (internal or external)

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. (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 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