Industrial Microbiology

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Industrial Microbiology INDM 4005 Lecture
10 24/02/04
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4. INFLUENCE OF PROCESS VARIABLES

• Overview
• Nutrient Limitation
• Cell Immobilisation

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Overview
4. Influence of process variables 4.1. Kinetics
and technology of nutrient limitation 4.1.1. Types
of continuous culture 4.1.2. Kinetics of
continuous culture 4.1.3. Typical pattern of
biomass and substrate levels in continuous
culture fermenter 4.1.4. Influence of growth
constants on biomass behaviour in continuous
culture 4.1.5. Application of continuous
culture 4.1.6. Advantages / disadvantages of
continuous culture 4.1.7. Modifications of basic
chemostat 4.2. Nutrient limitation also applied
in fed-batch 4.2.1. Fed-batch 4.2.2. Industrial
application of fed-batch 4.3. Nutrient
limitation and cell composition 4.4. Use of
continuous culture for calculation of growth
kinetics
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Batch Cultures

• Closed systems microorganisms undergo a
  predictable pattern of growth characterised by 4
  phases
• Describe the 4 phases of growth and the factors
  influencing them
• Understand the mathematics of exponential growth
• Define and apply growth parameters (td, m, mmax,
  k, Ys)
• Describe the Monod relationship and the meaning
  of ks

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4.1. KINETICS AND TECHNOLOGY OF NUTRIENT
LIMITATION

• Type of culture
• ? Batch ? varies during culture cycle
• ? Fed-batch ? is controlled or regulated after
  a certain time
• ? Continuous ? is controlled
• ? reflects the physiological state or
  intracellular environment
• ? control ? ? control intracellular environment

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Growth in Continuous Culture

• Scientists are trained to conduct experiments in
  which only one variable is changed at any one
  time
• Continuous culture methods enable constant cell
  numbers to be maintained in a constant chemical
  environment at specified growth rates for
  prolonged periods of time
• In this lecture we will focus on the theoretical
  and practical aspects of growth in flow-through
  systems

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Set up for Continuous culture
Fresh medium from reservoir Sterile air
Flow-rate regulator
Stirrer
Culture
Overflow Effluent
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4.1.1. TYPES OF CONTINUOUS CULTURE

• Method of control
• ? Chemostat - regulated by control of
  concentration of limiting nutrient
• ? Turbidostat - regulated by biomass using
  optical density (photoelectric cell)
• ? Biostat - regulated by systems monitoring
  biomass other than optical density (e.g CO2
  production)

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How can the population density and growth rate be
controlled?

• To regulate the growth rate and density it is
  necessary to control the influx of nutrients per
  unit time
• A distinctive feature of a chemostat is that one
  nutrient (C, N, P, energy source, growth factor)
  is at a low conc
• By selecting the concentration of substrate we
  can predetermine a certain microbial growth rate
• After a period of adjustment a steady-state
  equilibrium is achieved
• Changing the initial substrate concentrations
  alters the population density but growth rate
  remains unaltered at the new steady-state

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Fermenter configuration

• ? STR
• ? Up-flow
• ? Plugflow
• ? Single-stage
• ? Multi-stage
• ? Cell recycle
• Draw diagrams and make notes on the above

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• CASE STUDY
• Re Continuous Culture draw a diagram of a typical
  pilot/ laboratory system and an industrial system

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The development of growth in a chemostat
Steady State
Growth rate equals loss of cell biomass
Cell Number
Nutrient limitation causes decrease in m
Population density increases
mmax
Inoculation
Time in Hours
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Mathematical relationships of growth in chemostats

• Relationship between growth rate or specific
  growth rate and medium flow can be described
  mathematically
• The medium flow through the system is represented
  by the term dilution rate
• D
• D dilution rate (h-1)
• V culture volume (l)
• F flow rate (l h-1)

F V
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4.1.2. KINETICS OF CONTINUOUS CULTURE

• Thus
• Mass balance or the rate of change of cells in
  reactor RATE of ACCUMULATION minus RATE of
  LOSS
• dX /dt ?.X - D.x
• ? Mass balance of the substrate INPUT minus
  LOSS DUE TO OUTFLOW minus SUBSTRATE USED BY
  BIOMASS
• dS / dt D. Sr - D. S - ?. X / Y

X Total biomass D Dilution rate x Biomass
concentration m Specific growth rate Y
Yield S Substrate conc in fermenter Sr
Substrate conc in reservoir
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INCORPORATE MONOD MODEL

• The empirically derived equation for the
  relationship between specific growth rate and S
  is Monod equation
• D ? max . S / (Ks S)
• This is the most basic model for continuous
  culture
• NOTE When dX / dt 0 (at steady state) then
  D ?
• This equation demonstrates how the steady state
  substrate concentration in the chemostat is
  determined by the dilution rate

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Batch versus Chemostat
Exponential phase Chemostat of batch
culture operating in steady-state Growth
rate of culture Specific growth rate of
culture Biomass Available nutrients Culture
volume Toxic metabolites Constant, Variable,
Increasing, Decreasing
Increasing
Constant
Constant
Constant
Increasing
Constant
Decreasing
Constant
Constant
Constant
Increasing
Constant
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• CASE STUDY
• A chemostat operating in steady-state at a
  dilution rate of 0.25 h-1 sets a limiting
  nutrient concentration of 0.6 micromoles l-1.
  Determine the Monod constant in suitable units if
  mmax for the organism is 0.25 h-1

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D ? max . S / (Ks S) Rearrange the
equation ? max - D Ks s
D (0.6 - 0.25) Ks
0.6 0.25 Ks 0.6 x 1.4 Ks 0.84
micromoles l-1
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• THE PERFECT MODEL WOULD REQUIRE AN UNREALISTIC
  AMOUNT OF INFORMATION
• ? Simplifying assumptions are made, for example,
• ? Assume that population density has no effect
• ? If D 0 then batch culture - but no lag period
  predicted
• ? Transient conditions ? predicts either stable
  condition or wash-out
• ? Assumes all substrate goes to biomass
  (maintenance!)
• ? No allowance for substrate or product
  inhibition
• In more advanced models these areas must be
  considered

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4.1.3. TYPICAL PATTERN OF BIOMASS AND SUBSTRATE
LEVELS IN CONTINUOUS CULTURE FERMENTER

• CASE STUDY
• Plot
• ? steady state substrate concentration
• ? steady state biomass concentration
• ? steady state product concentration
• against dilution rate (?)
• Page 15 Stanbury Whitaker

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4.1.4. INFLUENCE OF GROWTH CONSTANTS ON BIOMASS

• BEHAVIOUR IN CONTINUOUS CULTURE
• ? Influence of low vs high Ks or ?max on biomass
  or substrate level
• ? Influence of low vs high Ks or ?max of
  different populations on competition
• DEVIATIONS FROM IDEAL BEHAVIOUR may be due to
• ? Maintenance energy
• ? Synthesis of reserve polymers
• ? Switch to less efficient pathways
• ? Imperfect mixing
• ? Substrate toxicity
• ? Second substrate becomes limiting

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4.1.5. APPLICATION OF CONTINUOUS CULTURE

• INDUSTRY
• Waste-treatment
• Single-cell protein
• Continuous beer production
• Continuous amino acids, organic acids production
• Continuous ethanol
• Continuous bakers yeast

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• RESEARCH - more important
• Physiology and biochemistry growth rate control
• Influence of environmental / process factors on
  growth and product formation.
• Induction, repression, growth rate, influence
  of temperature, pH etc.
• Microbial ecology
• Selection of slow growing populations
• Prey-predator interactions
• Competition (e.g plasmid /-)
• Kinetics
• Calculation of growth constants, fermentation
  data

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• CASE STUDY
• From the literature record some applications of
  continuous culture to studies in microbial
  physiology and ecology

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4.1.6 ADVANTAGES / DISADVANTAGES OF CC

• Advantages
• Uniformity of operation
• Process demands are constant
• i.e. continuous cycle of sterilisation,
  fermentation, harvesting, extraction
• Once in steady-state demands re process control
  are constant
• i.e. oxygen demand
• Disadvantages
• Susceptibility to contamination
• Duration of run is longer ? increased chance of
  contamination
• Strain degeneration arising from large number of
  generations
• Contamination with "fitter" competitor e.g.
  lower Ks

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OBJECTIVES IN INDUSTRIAL APPLICATION?

• ? CONTINUOUS PROCESSING
• Advantage ?
• example beer ? Residence time of "pint" in
  brewery same.
• example waste-treatment ? definite advantage.
• ? EXERT PHYSIOLOGICAL CONTROL
• Can use fed-batch which is less demanding

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4.1.7. MODIFICATIONS OF BASIC CHEMOSTAT

• MULTI-STAGE
• Different environments or growth rates in the
  various reactors (e.g. 1st ? biomass, 2nd ?
  product)
• SINGLE STAGE WITH CELL RECYCLE
• Application in activated sludge waste-treatment
• Relationship between D and ? different when
  recycle used.
• EFFECT OF FEEDBACK
• 1. Increase biomass conc. in fermenter - lower
  in effluent
• 2. Decrease residual substrate
• 3. Maximise rate of product formation
• 4. Dcrit is increased - useful when substrate is
  dilute

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Chemostats in series
F1 SR
X1 S1 V1
FO2 SR2
X2 S2 V2
F2
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• CONTINUOUS CULTURE PRINCIPLES
• Also applied in
• UP-FLOW REACTORS (often with immobilised cells)
• PLUG-FLOW SYSTEMS

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4.2. NUTRIENT LIMITATION ALSO APPLIED IN
FED-BATCH

• 4.2.1 Fed-Batch
• Takes advantage of fact that residual substrate
  concentration may be maintained at very low
  levels
• Type of continuous culture but volume is not
  constant.
• Quasi-steady state achieved.

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CLASSIFICATION OF FED-BATCH OPERATION

• Without feed-back control - programmed
  feed-rate
• 1. Intermittent addition
• 2. Constant rate
• 3. Exponentially increased rate
• With feed-back control
• 1. Indirect feed-back
• e.g. respiration rate, dissolved oxygen, pH
• 2. Direct feed-back
• concentration of substrate in culture exerts
  control

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4.2.2 INDUSTRIAL APPLICATION OF FED-BATCH

• Penicillin
• Glucose, phenyl acetic acid, ammonia source
• Cephalosporin
• Glucose, methionine
• Streptomycin
• Glucose, ammonia source
• Glutamic acid
• Urea, ethanol, (acetic acid)
• Amylase
• Carbon source
• Bakers Yeast
• Glucose
• Citric acid
• Glucose, ammonia

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4.3 NUTRIENT LIMITATION and CELL COMPOSITION

• Media can be designed to allow limitation on any
  essential nutrient
• NUTRIENT LIMITATION EFFECT
• CARBON ? energy supply
• NITROGEN or SULPHUR ? protein synthesis
• PHOSPHORUS ? Nucleic acid synthesis
• MAGNESIUM or POTASSIUM ? Nucleic acid and or
  membrane synthesis

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4.3 NUTRIENT LIMITATION and CELL COMPOSITION

• THE DEGREE OF LIMITATION INFLUENCES THE
• CELL COMPOSITION, for example
• ? CELL SIZE
• ? NUCLEIC ACIDS
• CONSEQUENTLY CELLS BEHAVE DIFFERENTLY UNDER
  DIFFERENT LIMITATION CONDITIONS
• Repression mechanisms may be removed, for
  example, antibiotic production or pigment
  production under phosphate limitation

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4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF
GROWTH KINETICS

• (1) Calculation of Ks and ?max
• (2) Determine variation in yield with growth rate
• (3) Calculation of Yg and m, endogenous
  respiration
• (4) ? /?max to compare growth under different
  conditions
• NOTE growth rate becomes an independent variable
  in continuous culture

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4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF
GROWTH KINETICS

• Use of higher dilution rates can lead to higher
  biomass productivity
• But result in
• higher substrate concentrations in the effluent
  and lower biomass concentrations in the reactor
  due to wash-out
• when the dilution rate exceeds the critical
  dilution rate then washout occurs

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4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF
GROWTH KINETICS

• These factors have a number of consequences e.g
  in continuous wastewater treatment processes
• The minimum reactor volume is set by the critical
  dilution rate
• High dilution rates will lead to an effluent
  containing high concentration of substrate and
  the effluent will therefore contain
  substrates/wastes and not have been treated
  properly
• Low cell concentrations at high dilution rates
  will also make the reactor sensitive to
  inhibitors in the feed. Inhibitors would cause
  the specific growth rate of the cells to drop and
  cause the cells to washout. The lower the conc of
  cells, then the faster the cells will washout

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Conclusions

• In this lecture we have seen that a chemostat is
  a means of providing nutrient limitation an
  important process variable
• Mathematical relationships can be used to
  predict growth and determine growth parameters
  such as mmax, Ks, Y
• List the differences between growth in batch and
  in continuous culture
• Understand the terms steady-state, dilution
  rate, growth limiting substrate, Monod constant,
• Describe the principles of fed-batch, biomass
  feedback, and multi-stage cultivation
• Give applications for continuous cultivation
  techniques
• Describe the main practical problems encountered
  in chemostat operation