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Microbial Growth
KineticsChapter 2
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Introduction Growth of microbes are results of:
- replication- change in cell size
Can grow under various physical, chemical andnutritional conditions. Convert nutrient from medium into biological
compounds
substrates + cells product + more cells Rate of growth cell concentration
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FermentationTraditionally-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
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Types of Processes Batch: (A)
Continuous: (B)
Fed-batch: (C)
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"closed system". Nothing is added exceptoxygen (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 atthe end of the process, optimization of cultural
conditions supporting growth should be established.
- For primary metabolite production, conditions toextend the exponential phase (and hence, product
excretion) should be provided
Batch Culture
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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
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Control systems for batch fermentation arenormally associated with pH, dissolved oxygen
and temperature.
In batch fermentation, if growth is subject tosubstrate inhibition, fermentation has to be
started with low initial substrate
concentration.
This result in lower maximum biomass andhence, probably, lower maximum
concentration of the required product.
Batch Culture
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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 betweendead/live cells
not suitable for aggregated cultures
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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 foryeast and bacteria
- selection for best medium growth is crucial
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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
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Cont..
4. Light intensity measurement
- Intensity of light depicts cell concentration- only for diluted suspension
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Determining Cell MassConcentration
Direct method:- Dry weight - biomass determination
- OD spectrophotometer
Indirect method:
- measurement of cellular component
- e.g: enzyme, chlorophyl
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Growth Patterns & Kinetics in Batch*
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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. Thecomposition of the culture
medium, the biomass
concentration, and the
metabolite concentration
generally change constantly
as a result of the metabolismof the cells.
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Cell growth kinetics
Batch culture
Growth rate of bacterial culture (duringexponential phase):
xdt
dx
1,
,
.
=
==
hrategrowthspecific
hrtimet
concbiomassx
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Growth-limiting Nutrient
Nutrient will be exhausted before theothers
Decrease in growth rate due to depletion ofsubstrate may be described using Monod
equation:
sK
s
s+
=max
= specific growth rate coeff.
max = max specific growth ratecoeff.
S = conc. of limiting nutrient
Ks = half saturation coeff.
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Lag Phase
Delay before rapid growth Occurimmediately 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
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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 growthall component growth at same rate
Growth rate independent of nutrient concentration
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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
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Stationary Phase
Resting phase Zero growth rate (no cell division) orgrowth rate
equal to the death rate
Nutrients depleted
Oxygen may be limited Release of cellular chemicals e.g. toxins
Cell growth~= cell death
Production ofsecondary metabolites (non growth
related). E.g: antibiotics, hormones Mixed growth and non-growth associated production.
Cell lysis, cryptic growth occur
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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 ordays.
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Product Formation
Primary metabolites
- Growth associated
Secondary metabolites
- Stationary growth associated
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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
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Secondary Metabolites
Formed at the end or during stationary phase
Not essential for growth
Growth conditions crucial to determine thesynthesis rate of secondary metabolites
Over-production often achievable (not growth
related)
Unpredictable - formation is not consistentamong all members of a species
Ex: penicillin, antibiotics.
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Microbial Product
Microbial growth, product formation andsubstrate utilization rates are usually
expressed in the form of specific rates
Classified to 3 categories:
growth associated
non growth associated
mixed growth associated
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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
h i d d
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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)
i d G h i d
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Mixed Growth Associated
Production occur during slow growth andstationary phase
E. g: lactic acid & certain secondary
metabolites
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Effect of Environmental FactorsOn Growth
Temperature
pH
Oxygen availability
Osmotic pressure/salt concentration
Nutrient availability
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Feed
medium
reservoir
Outflow
containing
product
Fi
FoProductivity = (F/V) x Product
concentration in outflow
V
CONTINUOUS CULTURE
An open system is set up. Sterile nutrient solution isadded to the bioreactor continuously and anequivalent amount of converted nutrient solution withmicroorganisms is simultaneously taken out of thesystem.
C ti C lt
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Continuous Culture
The importance of continuous culture:- maintenance of a culture in constantenvironmental conditions through continuoussupply 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 environmentalon cell physiology
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Continuous Culture
Fresh growth mediumis added continuously
during fermentationand spent medium isremoved.
Fermentation can lastup to 1,000 hours.
ow e row n ont nuous
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ow e row n ont nuousCulture
Fresh medium continually supplied to well-stirredculture
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
B h C i C l
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Batch vs. Continuous Culture
Batch culture; the culture environment continuouslychanges
Growth, product formation and substrate utilizationall terminate after a certain time interval
Continuous culture: fresh nutrient medium iscontinually 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
C t
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Cont
Provides constant environmental
conditions for growth and product formation
and supplies uniform quality product
For growth associated products has higherproductivity than batch culture
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Why is Batch Culture Predominantly Used in
the Biotech Industry
Many secondary products are not growth
associated
Genetic instability
Reliability
Economic considerations
D i f C ti C lt
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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 varywith axial position in the reactor
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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 cellsuspension 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 andcell concentrations are constant
- volume of reactor constant
Chemostat
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Chemostat
Effluent
Culture medium
Reservoir: Oneessential nutrientin growth-limitingamount
Flowregulator
Valve
Cont
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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
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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 atthe beginning of the fermentation and these
substances continue to be added in small
doses during the production phase.
F d B t h C lt
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Fed-Batch Culture
Batch culture which are fed continuously or sequentiallywith medium without the removal of culture fluid
Established initially in batch mode and is then fedaccordingly to one of the following feed strategies:
the same medium used to establish the batch culture isadded, resulting in an increase in volume
a solution of the limiting substrate at the sameconcentration as that in the initial medium is added,resulting in an increase in volume
a concentrated solution of the limiting substrate is addedat a rate less than in (i) and (ii), resulting in an increasein volume
a very concentrated solution of the limiting substrate isadded at a rate less than in (i), (ii) and (iii), resulting inan insignificant increase in volume
C t
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Cont
strategies (i) and (ii) - variable volume
strategy (iv) - fixed volume
strategy (iii) - culture intermediate between thetwo extremes of variable and fixed volume
Ex:
- production of bakers yeast
- formation of ethanol- production of hepatitis B surface antigen
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Limiting substrate is fed without diluting the culture.
Culture volume can be maintained constant by feedinggrowth-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 aphotosynthetic culture, radiation can be the growthlimiting factor without affecting culture volume.
1. Fixed volume fed-batch
2. Variable volume fed-batch
Volume changes with fermentation time due tosubstrate feed.
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