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Examples of Irreversible Inhibitors

diisopropylphosphofluoridate
prototype for the nerve gas sarin
permanently inactivates serine proteases by
forming a covalent bond with the active site
serine

Penicillin is a suicide inhibitor

Glycopeptide transpeptidase catalyzes the formation of cross-links


between D-amino acids in the cell walls of bacteria. This enzyme also
catalyzes the reverse reaction, the hydrolysis of peptide bonds. During
the course of hydrolyzing the strained peptide bond in penicillin, the
enzyme activates the inhibitor (penicillin), which then covalently
modifies an active site serine in the enzyme. In effect, the enzyme
commits suicide by hydrolyzing the strained peptide bond in
penicillin.

Suicide inhibitors work by


tricking the enzyme into
activating the inhibitor, which then
forms a covalent bond with the
enzyme, leading to its permanent
inactivation.

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MICROBIAL GROWTH
KINETICS

TYPE OF MICROBIAL GROWTH


SYSTEM

3 major type:
Batch culture
Continuous or Chemostat Culture
Fed-batch Culture

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BATCH
CULTURE

BATCH CULTURE

Definition: A closed system with limited amount of nutrient (no


additional of medium added)
Cells grows through several phases:
i) Lag phase
ii) Log phase
iii) Deceleration
phase
iv) Stationary
phase
v) Death phase

i)Time
Lag
phase
of adaptation of cell to the environment or medium

(reorganization of micro molecular contituents).


Length of lag phase may vary (depend on specific circumstances).
Shorter lag time is recommended for industry, can be achieved using
suitable inoculum (active or not) and environmental condition.
Synthesis or inhibition of the enzyme or cell structure components may
occur.
Typical effects:
Low cell number / cell concentration.
No changes in substrate pH.
No changes in substrate concentration.
No product formation.

ii) Log phase

Illustrate by a linear line of the plot of log cell mass vs time.


During this phase, a growth at steady state where specific
growth rate, are fixed.
Cell growth at maximum attainable rate.
Typical effects in log phase:
Rapid increase in cell concentration.
Rapid changes in substrate pH.
Substrate concentration decreased.
Product formation starts.

iii) Deceleration phase

Growth rate slowly decreases due to:


Consumption of nutrient or essential nutrient
become depleted (substrate limitation).
Accumulation of toxic product.

iv) Stationary phase

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iv) Death phase

GROWTH VS
MULTIPLICATIO
N CURVE

COMPARISON BETWEEN GROWTH AND


MULTIPLICATION CURVE
GROWTH CURVE

MULTIPLICATION CURVE

A plot of biomass concentration against


Incubation time.

A plot of cell number against time.

Lag phase is shorter than the multiplication


curve since the growth rate begins to
increase earlier than the multiplication rate.

Lag phase longer.

The log phase is substantially longest.

Shorter log phase.

Section 3 of both curve are identical.

Section 3 of both curve are identical.

Death phase sets in earlier.

Death phase sets in later.

Stationary phase is much longer.

Shorter stationary phase.

GROWTH CURVE

X = Biomass
concentration
Individual phases:
1: Lag phase
2: Accelaration
phase
3: Balanced growth
4: Deceleration
phase
5: Stationary phase
6: Death phase

MULTIPLICATION CURVE

N = No of cell
Individual phases:
1: Lag phase
2: Accelaration
phase
3: Balanced growth
4: Deceleration
phase
5: Stationary phase
6: Death phase

PRODUCTION
KINETICS

To determine the metabolic parameters

Need data on:


substrate uptake with time
with and without product formation
product generation with time
with and without cell growth
cell growth with time

Specific growth rate:

Where:
dx = Change in biomass concentration.
dt = Change in incubation time.
x = biomass concentration.
Specific growth rate, expressed in reciprocal time unit (h-1).

During batch cultivation, specific growth rate changes


continuosly from zero to the max value max.
max depends on microorganisms, physical, chemical
conditions.
Typical values of max:

Microorganisms

max (h-1)

Bacteria

Cultivation
Temperature
37C

Yeast

30C

0.3-0.5

Actinomycetes

28C

0.1-0.3

Fungal

28C

0.1-0.3

0.6-1.2

By plotting the growth curve of the microorganisms, then determine


the instavenous value at each sampling time by ascertaining the
tangent at the point of contact on the growth curve.
The highest value obtained (from 24-72h) is the max.

The Yield Coefficient (Y)

A measure of the overall efficiency of the conversion of


substrate to cell mass or specific product:

Parameter

Equation

Cell (Y x/s)

X / S

Product (Y p/s)

P / S

p/x)
/ X
Y isProduct
not (Y
constant,
will varyPdepending
on organism, pH,
temperature and substrate

Substrate Utilization and Product


Formation (Yp/s)
YP/S =
=

g/l product produced


g/g carbon sources utilized
g/g

Economic yield (Yp/x)


YP/X =
=

g/l product produced


g/L biomass formed
g/g

Batch Productivity

Productivity a measure of product ( or biomass)


produced per unit time (g/L/h).
Product formation of growth link product is
closely related with growth rate.
Productivity in batch culture will be a greatest
when growth rate max (max).

Productivity
(R batch) =

X max Xo
T final T
initial

Where;
X max = maximum cell concentration at stationary phase
Xo = initial cell during inoculation
T final = time during which organism growing at max
T initial = time which organism not growing at max, including lag phase,
deceleration phase period of batching, sterilizing and so on.

Incubatio dt
n Time
(h)

1/t

[Cell] (g/L)

1/x

dx/dt

(h-1)

0.1

0.004

0.2

10.0

0.004

0.04

0.3

5.0

0.004

0.021

0.3

3.33

0
24

0.042

24

0.1
48

0.021

48

0.2
72

72

0.014
0.3

96
96

0.3
120

120

0.2
144

144

0.1
168

168

0.05
192

192

dx

0.02

CONTINUOUS
CULTURE

CONTINUOUS CULTURE

Fresh fermentation media is continuosly added to the reactor


while fermenter broth containing biomass, products and unused
nutrient are continuosly removed.
Exponential growth in batch culture may be prolonged by the
addition of fresh medium to the vessel.
Growth can be maintained for long duration
Continuous feeding to a culture at a suitable rate
formation of new biomass by the culture is balanced by the loss
of cell from the vessel
STEADY STATE.

Application of Continuous Culture

Biomass production
Growth associated product or primary metabolite e.g: ethanol,
citric acid
Not suitable for non-growth associated or secondary metabolite
e.g: antibiotic

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Important

When referring to continuous culture systems, the terms


used in batch culture (lag, exponential, stationary, death
phase)
have no meaning because the system is operating
continuously and growth cannot segregated into phases

BATCH CULTURE

CONTINUOUS CULTURE

Nutrients added only at start

Nutrients added continuously

Product removed when fermentation stops.

Product continuously removed .

Growth rates and product formation are slower


because limiting factors
ex: substrate levels/ build up of toxins.

Organism held in exponential growth phase giving


higher productivity so can be on a smaller scale.

Slower growth rates = Larger vessels are used.


Easy to set up and maintain.

Can be very difficult to maintain conditions so that


exponential phase is maintained. Foaming,
clumping and blocked inlet pose problems.

If contamination occurs only one batch is


wasted.

Contamination can afferct a huge volume of


product/ organism.

Less efficient / more time wasted shutting down


removing product and starting up again.

Continuous, therefore more efficient use of time.

Product quality can vary between batches.

Product quality more consistent.

FED-BATCH
CULTURE

FED BATCH CULTURE

Extending the batch culture by feeding


continuously or periodically with
medium with no removal of culture from
the vessel.
Somewhere
between
batch
and
continuous culture.
A volume of medium is inoculated with
the organism and allowed to grow for a
batch period of time.
Subsequently, a feed is initiated into the
fermenter when a quasi steady state is
obtained.
Quasi steady state: when the growth
limiting substrate has depleted.

PRODUCT
FORMATION

Production kinetics

Classified based on the relationship between product


synthesis and energy generation in the cell
Growth associated.
Non-growth associated.
Mixed-growth associated.

Products

Growth-associated.
produced at the same time as cell growth.
constitutive enzymes (ones that are normally present).
glucose isomerase.
metabolic intermediates.
pyruvate, citrate, acetate.
Non-growth-associated.
takes place during the stationary phase (m=0)
secondary metabolites.
Antibiotics.
Mixed - growth associated / Partially growth associated.
takes place during growth and stationary phases.
metabolic by-products.
lactate, ethanol.
secondary metabolites.

Primary Metabolite

Released as a result of metabolic processes which are essential for the


life of the micro-organism e.g. ethanol from Saccharomyces
cerevisiae.
Thus, primary metabolites are produced throughout the growth of the
micro-organism, especially through the exponential phase.

Secondary Metabolite

A substance which is not essential for the life of the micro-organisms


and which is not produced as a result of the growth process e.g.
penicillin.
Secondary metabolites are produced after the exponential growth
phase has stopped.
This is important because it means that secondary metabolites such as
penicillin cannot be produced in continuous fermenters which
deliberately maintain the micro-organism in the exponential growth
stage.

Production of secondary metabolite starts when exponential growth stops


and growth of cells starts to slow.
Adding a lot of extra nutrients at time T (see graph) will simply increase the
growth of the micro-organism but not formation of the product.
However, by adding a small amount of extra nutrients at this time, the
amount of product formed can be increased.