Microbial Growth

Kinetics
Chapter 2
 Introduction
• Growth of microbes are results of:
- replication
- change in cell size
• Can grow under various physical, chemical and
nutritional conditions.
• Convert nutrient from medium into biological
compounds

substrates + cells product + more cells

• Rate of growth ∞ cell concentration
 Fermentation
• Traditionally-the process for the production
of alcohol or lactic acid from glucose
• Broadly-an enzymatically controlled
transformation of organic compound.
• Fermentation may be carried out as:
 Batch process
 Continuous process
 Fed-batch process
 Types of Processes
• Batch: (A)
• Continuous: (B)
• Fed-batch: (C)
 Batch Culture
 "closed system". Nothing is added except
oxygen (in the form of air), an antifoam agent,
and acid or base (control pH).
 - used to produce biomass, primary metabolites
- In order to produce maximum possible biomass at
the end of the process, optimization of cultural
conditions supporting growth should be established.

- For primary metabolite production, conditions to

extend the exponential phase (and hence, product
excretion) should be provided
 Batch Culture
Principal advantages of batch cultures are;

low contamination risk;

the ability to run different succesive phases in the
same vessel;
and close control of the genetic stability of the
microorganism
 Batch Culture

Control systems for batch fermentation are

normally associated with pH, dissolved oxygen
and temperature.

In batch fermentation, if growth is subject to

substrate inhibition, fermentation has to be
started with low initial substrate
concentration.

This result in lower maximum biomass and

hence, probably, lower maximum
concentration of the required product.
 Batch Culture

• Culturing a cell without further

addition or removal of nutrient
• Cell must be quantified either:
 directly- due to presence of
suspended solids
Indirectly (cell number, cell mass)
 Determining Cell Density Number

1. Petroff-Hausser slide/hemocytometer
- 20 grid squares in counted using
microscope-average
- Disadvantages:
 medium must be free from particles
 stain is used to differentiate between
dead/live cells
 not suitable for aggregated cultures
 Cont..

2. Plate count:
- used for counting viable cell
- unit: colony forming unit (CFU)
- cultures are diluted and pipetted or spread on
agar surfaces
- plates are incubated and viable colonies are
counted
- a good plate count must consist between 30-
200 colonies
- suitable for yeast and bacteria
- selection for best medium growth is crucial
 Cont..

3. Electrical resistance of cells

- cells pass the orifice causes resistance
and provide pulses
- number pulses is a measure - number of
cells
- height of pulses- measure cell size
 Cont..

4. Light intensity measurement

- Intensity of light depicts cell concentration
- only for diluted suspension
 Determining Cell Mass
Concentration
• Direct method:
- Dry weight - biomass determination
- OD – spectrophotometer

• Indirect method:
- measurement of cellular component
- e.g: enzyme, chlorophyl
Growth Patterns & Kinetics in Batch*
 The fermenter could be operated in different modes aims at 
improving the performance.
BATCH FERMENTATION
Considered to be a closed
system. At time t=0 the
sterilized nutrient solution in
the fermenter is inoculated
with microorganisms and
incubation is allowed to
proceed. In the course of the
entire fermentation, nothing
is added, except oxygen (in
case of aerobic
microorganisms), an antifoam
agent, and acid or base to
control the pH. The
composition of the culture
medium, the biomass
concentration, and the
metabolite concentration
generally change constantly
as a result of the metabolism
of the cells.
 Cell growth kinetics
• Batch culture
• Growth rate of bacterial culture (during
exponential phase):

dx
= µx
dt
x = biomass conc.
t = time, hr
µ = specific growth rate, h −1
 Growth-limiting Nutrient
• Nutrient will be exhausted before the
others
• Decrease in growth rate due to depletion of
substrate may be described using Monod
equation: s
µ = µ max
Ks + s
µ = specific growth rate coeff.
µmax = max specific growth rate
coeff.
S = conc. of limiting nutrient
Ks = half saturation coeff.
 Lag Phase
• Delay before rapid growth
• Occur immediately after inoculation
• Cell mass increase, number of cells remain
constant
• Cells may be damaged
• Cells may be adapting to media
• Cells may be old/cold
• Cells make new ribosomes
• Cells make new proteins
• Cells begin to make cells
 Log/Exponential Phase
• Cells divide at or close to maximum
• Cell adjusted to new environment
• Biomass increases quickly
• Nutrients consumed rapidly
• Oxygen (if needed) consumed rapidly
• Heat produced in some cultures
• Changes in pH due to organism
• Protein in media may produce foam
• Cell mass & numbers multiply rapidly
• Balance growthall component growth at same rate
• Growth rate independent of nutrient concentration
 Deceleration Phase

• Also known as “late log phase”

• Growth decelerates due to:
- depletion of essential nutrient
- accumulation of toxic by product
- unbalanced growth-restructuring of cell
 Stationary Phase

• Resting phase
• Zero growth rate (no cell division) or growth rate
equal to the death rate
• Nutrients depleted
• Oxygen may be limited
• Release of cellular chemicals e.g. toxins
• Cell growth~= cell death
• Production of secondary metabolites (non growth
related). E.g: antibiotics, hormones
• Mixed growth and non-growth associated production.
• Cell lysis, cryptic growth occur
 Death Phase

• Cells decline exponentially

• Some may start during stationary phase
• Cell autolysis may occur
• Occur at the end because of nutrient
depletion or waste accumulation
• Sometimes death rate change after hours or
days.
 Product Formation

• Primary metabolites
- Growth associated

• Secondary metabolites
- Stationary growth associated
 Primary Metabolites

• Formed during active cell growth

• During primary growth phase
• Product essential for the metabolic activity &
growth
• Produced from growth substrate by the cell
activity
• Ex: alcohol, amino acid
 Secondary Metabolites

• Formed at the end or during stationary phase

• Not essential for growth
• Growth conditions crucial to determine the
synthesis rate of secondary metabolites
• Over-production often achievable (not growth
related)
• Unpredictable - formation is not consistent
among all members of a species
• Ex: penicillin, antibiotics.
 Microbial Product

• Microbial growth, product formation and

substrate utilization rates are usually
expressed in the form of specific rates
• Classified to 3 categories:
 growth associated
 non growth associated
mixed growth associated
 Growth Associated Product

• Produced simultaneously with growth

• Specific rate product formation ∞ specific
growth rate
• Product: primary metabolites
• E.g.: Enzyme protease (Bacillus subtilis),
amino acid
 Non Growth Associated Product

• Production occur during stationary phase

• The specific rate of product formation is
constant
• Product: secondary metabolite
• E. g: hormones, antibiotics (penicillin)
 Mixed Growth Associated

• Production occur during slow growth and

stationary phase
• E. g: lactic acid & certain secondary
metabolites
 Effect of Environmental Factors
On Growth
• Temperature
• pH
• Oxygen availability
• Osmotic pressure/salt concentration
• Nutrient availability
CONTINUOUS CULTURE
 
An open system is set up. Sterile nutrient solution is
added to the bioreactor continuously and an
equivalent amount of converted nutrient solution with
microorganisms is simultaneously taken out of the
system.

Feed  Fi
medium 
reservoir V

Productivity = (F/V) x Product  Fo
concentration in outflow Outflow 
containing 
product
 Continuous Culture

• The importance of continuous culture:

- maintenance of a culture in constant
environmental conditions through continuous
supply of nutrient
- provision of nutrients and removal of wastes.
- useful for:
 study in a certain growth phase
 evolution studies
 study the effect of changes in the environmental
on cell physiology
 Continuous Culture

Fresh growth medium

is added continuously
during fermentation
and spent medium is
removed.

Fermentation can last

up to 1,000 hours.
 How Cell Grow in Continuous
Culture
• Fresh medium continually supplied to well-stirred
culture
• Product (cell/culture medium) continuously
withdrawn
• During cultivation, growth & product formation
can be prolonged
• At steady state: cell, product and substrate
concentration remain constant
• An essential nutrient is in limiting quantities
 Batch vs. Continuous Culture

• Batch culture; the culture environment continuously

changes
• Growth, product formation and substrate utilization
all terminate after a certain time interval
• Continuous culture: fresh nutrient medium is
continually supplied to a well-mixed culture,
products and cells are simultaneously withdrawn
• Growth and product formation can be maintained for
prolonged periods of time
• At steady state, cell, product and substrate
concentrations remain constant
 Cont…

• Provides constant environmental

conditions for growth and product formation
and supplies uniform quality product
• For growth associated products has higher
productivity than batch culture
Why is Batch Culture Predominantly Used in
the Biotech Industry

• Many secondary products are not growth

associated
• Genetic instability
• Reliability
• Economic considerations
 Devices for Continuous Culture

Plug flow reactor (PFR)

- Continuous cultivation
- No back mixing-fluid elements containing
active cells cannot inoculate other fluid
elements at different axial positions
- Substrate and cell concentrations vary
with axial position in the reactor
 Cont…
 Chemostat
- also known as a continuous stirred tank reactor (CSTR)
- refers to constant chemical environment
- perfectly mixed continuous flow
- equipped with pH, DO, level controller
- feeding of fresh medium and removal of cell
suspension occur at the same rate
- cellular growth is typically limited by one essential
nutrient: other nutrients are in excess
- when operated at steady state: nutrient, product and
cell concentrations are constant
- volume of reactor constant
 Chemostat
Culture medium
Reservoir: One
essential nutrient Flow
in growth-limiting regulator
amount

Valve

Effluent
 Cont…
 Turbidostat
- Cell concentration in the culture vessel
constant (monitor the OD & feed flow rate)
- Volume is kept constantly by removal of culture
broth
- Suitable for microorganisms able to withstand
environmental stress
- Flow rate into the system is adjusted to
maintain preset turbidity (cell density)
- No limiting nutrients
- Operates best at high dilution rates
FED BATCH FERMENTATION
Substrate is added in increments as the
fermentation progresses. In the fed-batch
method the critical elements of the nutrient
solution are added in small concentrations at
the beginning of the fermentation and these
substances continue to be added in small
doses during the production phase.
 Fed-Batch Culture
• Batch culture which are fed continuously or sequentially
with medium without the removal of culture fluid
• Established initially in batch mode and is then fed
accordingly to one of the following feed strategies:
 the same medium used to establish the batch culture is
added, resulting in an increase in volume
 a solution of the limiting substrate at the same
concentration as that in the initial medium is added,
resulting in an increase in volume
 a concentrated solution of the limiting substrate is added
at a rate less than in (i) and (ii), resulting in an increase
in volume
 a very concentrated solution of the limiting substrate is
added at a rate less than in (i), (ii) and (iii), resulting in
an insignificant increase in volume
 Cont…

• strategies (i) and (ii) - variable volume

• strategy (iv) - fixed volume
• strategy (iii) - culture intermediate between the
two extremes of variable and fixed volume
• Ex:
- production of bakers’ yeast
- formation of ethanol
- production of hepatitis B surface antigen
  1. Fixed volume fed-
batch
 Limiting substrate is fed without diluting the culture.

Culture volume can be maintained constant by feeding

growth-limiting substrate in undiluted form, for example,
as a very concentrated liquid or gas (ex. Oxygen).
 Alternatively, substrate can be added by dialysis or, in a
photosynthetic culture, radiation can be the growth
limiting factor without affecting culture volume.

  2. Variable volume fed-batch

 Volume changes with fermentation time due to
substrate feed.