Beruflich Dokumente
Kultur Dokumente
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
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..
• 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 growthall component growth at same rate
• Growth rate independent of nutrient concentration
Deceleration 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
• Primary metabolites
- Growth associated
• Secondary metabolites
- Stationary growth associated
Primary Metabolites
Feed Fi
medium
reservoir V
Productivity = (F/V) x Product Fo
concentration in outflow Outflow
containing
product
Continuous Culture
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…