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CHAPTER 11

HOMOGENOUS REACTION

Cell growth kinetics


Growth kinetics in plasmid instability
Production kinetics in cell culture
Kinetics of substrate uptake in cell culture
Effect of culture conditions on cell kinetics
Determining cell kinetic parameters from batch data
Effect of maintenance on yields
Kinetics of cell death.

Growth Kinetics
Introduction
- Quantifying cell concentration:
- direct: no suspended solid and interference compounds.
cell mass concentration preferred dry weight, optical density
(600-700nm Wave Length)
cell number density: hemocytometer, plate counts, etc.

(turbidity)

-Quantifying cell concentration:


- indirect: direct method is inapplicable. (mold solid state fermentation)
Cell mass can be determined by measurement of protein, DNA or ATP. e.g. 1mg ATP/g
dry weight bacterial cell.

If 100 mg ATP/L is measured, then the cell mass:


100 mg (ATP/L)/1 mg ATP/g dry cells=100 (g dry weight cells/L)

Growth Kinetics: Batch Culture


-

All needed nutrients initially added


Products removed after a given period
(usually when growth is completed)
Nutrients and other components added Typical growth curve for a bacterial population
during growth are: gases, e.g. O2, titrants
for pH control, antifoam

phase
Lag phase

description

rate of growth is
zero; cell use to
adapt their new
environment
Acceleration phase growth rate start
Growth phase
Growth rate
achieve max
Decline phase
growth rate slow
due to nutrient
exhaustion or buid
up of inhibitory
product
Stationary phase
growth ceases
Death phase
cell lose viability
and lyse

Specific growth
rate
0

< max
max
< max

=0
< max

Batch Growth Kinetics


Lag phase
A period of adaptation for the cells to their
new environment
New enzymes are synthesized.

A slight increase in cell mass and


volume, but no increase in cell
number
Prolonged by low inoculum volume,
poor inoculum condition (high % of
dead cells), age of inoculum, nutrientpoor medium

Diauxic growth

Multiple lag phases: (diauxic


growth) medium contains more than
one carbon source

Typical growth curve for a bacterial population

Diauxic growth:
caused by a shift in metabolic pathways in the middle of a growth cycle

After once carbon source is exhausted, the cells adapt their metabolic
activities to utilize the second carbon source
The first carbon source is more readily utilizable than the second and
the presence of more readily available carbon represses the synthesis of
the enzymes required for the metabolism of the second substrate

Exponential growth phase


In this phase, the cells have adjusted to their new environment
and multiply rapidly (exponentially)
Balanced growth all components of a cell grow at the same rate.
During balanced growth, the net specific growth rate determined
from either cell number of cell mass would be the same
Growth rate is independent of nutrient concentration, as nutrients are
in excess.

The balance of cell mass in a batch culture gives:


dX
net X , X X 0 at t 0
dt
Integration of the aboveequation yields:
ln

X
nett , or X X 0e net t
X0

X and X 0 are cell concentrations at time t and t 0

Exponential growth phase


net R m
m is the maximum specific growth rate (1/time)
Doubling time of cell mass: the time required to double the microbial mass:

ln X / X 0

net

ln 2

net

0.693

net

Balanced growth

Exponential growth is balanced growth.


That is, all cellular constituents are manufactured at constant rates relative to
each other.
If nutrient levels or other environmental conditions change, unbalanced growth
results.
Specific rate of production of each component in culture must equal to cell
specific growth rate:

rz z

Where Z: cellular constituent (e.g. Protein, RNA, polysaccharide etc.)


rz: volumetric rate of production Z
z: concentration of Z in the reactor volume

Deceleration growth phase

Very short phase, during which growth decelerates due to either:


Depletion of one or more essential nutrients
The accumulation of toxic by-products of growth (e.g. Ethanol in yeast
fermentations)
Period of unbalanced growth: Cells undergo internal restructuring to increase
their chances of survival

Stationary Phase:
With the exhaustion of nutrients (S0) and build-up of waste and
secondary metabolic products
The growth rate equals the death rate.
There is no net growth in the organism population.
cells may have active metabolism to produce secondary metabolites.
Endogenous metabolism of energy stores can result in maintaining cell
viability

dX
k d X
dt
kd is the rate constant for endogenous metabolism.
The rate describing the conversion of cell mass into maintenance energy
or the loss of cell mass due to cell lysis:
Removal of inhibitory compounds will result in further growth if additional

substrate is provided
Primary metabolites are growth-related: ethanol by S. cerevisae.
Secondary metabolites are non-growth-related: antibiotics, pigments.

Death Phase:
The living organism population decreases with time, due to a lack of nutrients and
toxic metabolic by-products.
The rate of death usually follows:

dN
'
k d N
dt
'
kd is the first - order death rate constant.

Kinetic Pattern of Growth and Product Formation

Growth-associated

Mixed-growth-associated

Non growth-associated

Lactic acid fermentation, xanthan gum, and some secondary metabolites from cell
culture are examples of mixed-growth-associated products

11.7.3 Effect of substrate concentration


During the growth and decline phases of batch culture, the specific growth
rate of cell dependent on concentration of nutrient in a medium.
This component known as:
Growth limiting substrate : often carbon or nitrogen source in some case
oxygen or oxidant
Monod equation:
max

Ks s

Where
s = concentration of growth limiting substrate
max= maximum specific growth rate (T -1)
Ks = substrate constant

11.9 Production Kinetics in cell culture


Classified based on the relationship between product synthesis and energy

generation in the cell

Irrespective of the class of product, rate of product formation in cell


culture can be expressed as the function of biomass concentration:

rp q p x
Where rp= volumetric rate of formation (eg: kg m-3 s-1)
x = biomass concentration
qp= specific rate of product formation (T-1)

Case 1: Product formation directly coupled with energy


metabolism

rp YPX r x m p x
rp= volumetric rate of product formation (eg: kg m-3 s-1)
YPX= theoretical or true yield of product from biomass
rx= volumetric rate of biomass formation
mp= specific rate of product formation due to maintenance (T-1)
x= biomass concentration

Since

rx = x

rp (YPX m p ) x

From this equation, qp equal to a combination of growth associated and non


growth associated terms:

q p YPX m p

Case 2 : Product Formation Indirectly Coupled With Energy


Metabolism
Relationship between product formation and growth can be
complicated. Beyond the scope here.

Case 3 : Product Formation Not Coupled With Energy


Metabolism
Production not involving energy metabolism is difficult to relate to
growth because growth and product synthesis are dissociated.
Rate of formation of non-growth-associated product can be directly
proportional to biomass concentration,

constant qp

qp = complex function of growth rate


empirical equations derived from experiment.

11.10 Kinetics of Substrate Uptake in cell culture


Cells consume substrate from the external environment and channel it

into different metabolic pathway. Some substrate may be directed into


growth and product synthesis; another is used to generate energy for
maintenance activities.
Substrate requirements for maintenance vary considerably depending on

the organism and culture conditions.


The specific rate of substrate uptake for maintenance activities is known

as the maintenance coefficient, ms. Dimensions of ms are T-1, typical units


are kg substrate (kg biomass)-1 s-1.

Ionic strength greatly influences the value of ms.

physiological significance of ms for a particular organism


may not be constant at all possible growth rates.
Rate of substrate uptake can be expressed as a function
of biomass concentration by an equation analogous

Where,

rs = q s x

rs = volumetric rate of substrate consumption (kg m-3 s-1)


qs = specific rate of substrate uptake
x= biomass concentration

11.10.1 Substrate Uptake in the Absence of Product


Formation
In the absence of product formation, we assume that all substrate

entering the cell is used for growth and maintenance functions. Rates of
these cell activities are related as;

= r / Y + ms x

s
x
xs
Where,
rx = volumetric rate of biomass production
Yxs = true yield of biomass from substrate
ms = maintenance coefficient
x = biomass concentration

when rx is expressed using equation rx = x so the equation becomes


rs = - ( / Yxs + ms ) x

if we express as a function of substrate concentration:


rs = - [ maxs / Yxs (Ks + s) + ms ] x

(eq. 11.74)

when s is zero Eq. 11.74 predicts that substrate consumption will

proceed at a rate equal to ms x.

11.10.2 Substrate Uptake With Product Formation


Depend on whether product formation is coupled to energy metabolism.
When products are formed in energy-generating (i.e. anaerobic culture),

product synthesis is an unavoidable consequence of cell growth and


maintenance (Fig. 11.13(a))
No separate flow of substrate into the cell for product synthesis; product is
formed from the substrate taken up to support growth and maintenance.
Substrate consumed for maintenance does not contribute to growth; it
therefore constitutes a separate substrate flow into the cell.

In contrast, when production is not linked or only partly linked to

energy metabolism, all or some of the substrate required for


product synthesis is additional to, and separate from, that needed
for growth and maintenance (Fig 11.13 (b)).
When products are directly linked to energy generation, equations
for rate of substrate consumption do not include a separate term
for production; substrate requirement for product formation are
already accounted for in terms for growth and maintenanceassociated substrate uptake.

In culture where product synthesis is only indirectly coupled to energy

metabolism, rate of substrate consumption is a function of three factors: growth


rate, rate of product formation and rate of substrate uptake for maintenance.
These different cell functions can be related using yield and maintenance
coefficients;
rs = (rx / Yxs )+ (rp / Yps )+ ms x
Where,

rs = volumetric rate of substrate consumption


rx = volumetric rate of biomass production
rp = volumetric rate of product formation
Yxs = true yields of biomass from substrate
Yps = true yields of product from substrate
ms = maintenance coefficient
x = biomass concentration
We can express rx and rp using equation 11.52 and 11.67
rs = ( / Yxs + qp / Yps + ms) x

11.11 Effect of cell conditions on cell kinetics


By temperature:

- has a direct influence on reaction rate


- only have a minor effect on biomass yield coefficient, YXS .
By pH:
- effect on growth rate in much the same way as enzyme activity
- effect the profile of product synthesis in anaerobic culture
- can change maintenance energy requirement

11.12 Determining Cell Kinetic Parameters Batch


Data
In order to apply the equations presented (Sections 11.6-11.10 )to real

fermentations, we must know:


- the kinetic parameters for the system
- yield parameters for the system
- have information about rates of growth, substrate uptake and product
formation.
Batch culture is the most frequently-applied method for investigating

kinetic behavior, but it is not always the best.

11.13 Effect of Maintenance on Yields


It is difficult to evaluate the true yields e.g. Yxs, YPX and YPS
So, theoretical yields can be related to observed yields such as Yxs, YPX and

P YPS which are more easily determined.


11.13.1 Observed Yields

Expressions for observed yield coefficients can be obtained by applying Eq. (11.48):

YXS = -dX = rX
dS

(11.77)

rS

YPX = -dP = rP

(11.78)

dX rX
YPS = -dP = rP
dS
where

X, S , P

(11.79)

rS
= masses of cells, substrate and product

rX, rS, rp = observed rates evaluated from experimental data.

Yield coefficients can be determined by plotting X, S or P against each other

and evaluating the slope as illustrated in Figure 11.14.


Observed yield coefficients at a particular instant in time can be calculated as the
ratio of rates evaluated at that instant which are not necessarily constant
throughout batch culture.

11.13.2 Biomass Yield from Substrate


Equations for true biomass yield can be determined for systems without extracellular

product formation or when product synthesis is directly coupled to energy metabolism.


Substituting expressions for rX and rS from Eq. (11.52) and (11.73) into Eq. (11.77)

gives:
YXS = ____
[(/YXS) + mS]

(11.80)

Inverting the Eq. gave:


1/YXS = (1/ YXS) + (mS/)

(11.81)

If Yxs and mS are relatively constant, plot of l/Y'xs versus 1/, gives a straight line with

slope mS and intercept 1/Yxs.

YXS can be improved by decreasing the maintenance coefficient or increasing

the growth rate.

mS may be reduced by :

1. Lowering the temperature of fermentation, using a medium of lower ionic


strength
2. Applying a different organism or strain with lower maintenance
energy requirements.

11.13.3 Product Yield from Biomass


Observed yield of product from biomass YPX is defined in Eq. (11.78). When

product synthesis is directly coupled to energy metabolism, rP given by Eq.


(11.69).
Substituting this and Eq. (11.52) into Eq. (11.78) gives:
YPX = YPX + (mP/)
(11.82)
Extent of deviation of YPX from YPX depends on the relative magnitudes of mP

and .
To increase the observed yield of product, mP should be increased and
decreased.
Eq. (11.82) does not apply to products not directly coupled with energy
metabolism

11.13.4 Product Yield from Substrate


Observed product yield from substrate YP's is defined in Eq. (11.79). For

products coupled to energy generation, expressions for rp and rs are available


from Eqs (11.69) and (11.73). Therefore:
YPS = (YPX + mP) / [(/ YXS) + mS]

(11.83)

In many anaerobic fermentation i.e. ethanol production, yield of product

from substrate is a critical factor affecting process economics.


At high YPS, more ethanol is produced per mass of carbohydrate consumed

so that the cost of production is reduced.

Growth rate has a strong effect on YPS for ethanol;


Since YPS is low when = max,
So, it is desirable to reduce the specific growth rate of the cells.

Low growth rate can be obtained by:


Depriving the cells of some essential nutrient,

e.g. a nitrogen source,


Immobilizing the cells to prevent growth.

11.14 Kinetics of cell death


Is an important consideration in design of sterilization

process and in analysis of fermentations.


Cell death is assumed to be 1st order process:

rd kd N
Where,

rd = cell death
N = no of viable cell
kd = specific death constant

Rate of cell death can be expressed using cell concentration:

rd kd x

Where;

kd = specific death constant based on cell concentration


x = concentration of viable cell
In closed system, rate of cell death is equal to the rate of decrease in cell no.

If kd is constant, we can derive an

dN
rd
kd N
dt

expression of N as a function of time:

In N = In N0 - kdt