Fermentation Kinetics

Why DO We Want TO Know Fermentation Kinetics ?

• Overriding factor which propels biotechnology is PROFIT.

• Without profit - no money - no research and development and consequently no new

products.

• A biotechnologist role - to use biological systems to either maximize profits or

maximize the efficiency of resource utilization.
• Central aim of production fermentation - large scale cultivation of cells for
production of a large proportion of commercially important biological products.

• Obviously the maximization of profits is closely linked to optimizing product

formation by cellular catalysts; i.e. producing the maximum amount of product in
the shortest time at the lowest cost.
• To achieve this objective, the kinetics of the process must be known. In other
words, cell culture systems must be described quantitatively.
• Well understood kinetics of the system – helps in prediction of yields and reaction
times and correct size of a bioreactor.
• Much before the construction of full scale system, reaction kinetics must be
determined.
• In practice, kinetic data is obtained in small scale reactors and then used with mass
transfer data to scale-up the process.
Fermentation Kinetics - Mathematical Models
• Cell culture systems are extremely complex. There are many inputs and many
outputs.

• Unlike most chemical systems, the catalysts themselves are self propagating.
• To assist in both understanding quantifying cell culture systems, biotechnologists
often use mathematical models.

• A mathematical models is a mathematical description of a physical system. A good

mathematical model will focus on the important aspects of a particular process to
yield useful results.
Chemical Kinetic Equations as Mathematical Models
• Consider a first order chemical reaction in which 1 mole of reactant (S) is converted
to a product (P)

can be expressed as a differential equation of the form:

where [S] is the concentration of the reactant and k is a rate constant.

• For this reaction, a differential equation describing product formation is:

- or -

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 where [P] is the concentration of the product and n is the stoichiometric yield
constant describing the relationship between the removal of S and formation of P.
• Note that as the concentration of S decreases, the concentration of P increases and
hence the negative sign in equations 4 and 5.
• By solving these two equations, it is possible to predict the values of S and P at any
time.
Relation of Time, Substrate, Product
• Equation which relates substrate concentration and time
» ln [S2] - ln [S1] = - k(t2-t1)
Where k = rate constant

• Equation which relates substrate concentration and product formation

» [P2] = n ([S1] - [S2]) + [P1]
Where n = stoichiometric yield coefficient
Michaelis Menten Equation as a Mathematical Model
• In enzyme studies, you will have learnt the Michaelis Menten equation which is
mathematical model describing activity of many different enzymes -

Where V = rate of substrate removal

Vmax = maximum specific rate
Km = saturation constant
• The Michaelis Menten equation describes the rate of substrate breakdown by an
enzyme and can be written as a differential equation:

• Note that rate of formation of a product [P] from this reaction would be:
• or

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 where [P] = Concentration of the Product
Y = Stoichiometric Yield Coefficient.
• Mathematical models of cell culture systems have a very similar appearance to the
Michaelis Menten model.
Mathematical models - parameters, variables and constraints
• Several points should be noted from the above example of mathematical model

• The use of differential equation -

» Differential equations describe rates of change within a system.
» Many mathematical models are formulated using differential equations.
• Each equation contains variables and parameters. The variables in the Michaelis
Menten model are [S] and [P]. The values of variables will change with time.
• Vmax, Km and Y are assumed to be not changing with time. These expressions are
examples of parameters. Parameters are terms which are assumed to be constant
under a given set of conditions. With each different condition e.g. pH or
temperature, or a different calatalyst, a different set of parameters are required.
• There are many simplifying assumptions. For example, in the Michaelis Menten
model (equations 6-8), temperature and pH are assumed to remain constant. This
means that the model will apply only when these conditions OR ‘Parameters’ are
met.

• ‘Variables’ are expressed as concentrations (e.g. g.l-1) rather than as absolute

values (e.g. g). This is not obligatory but the use of relative expressions makes the
model more useful when used to scale-up a process.

• The model also assumes that the temperature is suitable for enzyme activity; The
temperature and pH range for which the model applies are examples of
‘constraints’. Constraints are the limits within which the model is valid.
Stoichiometric yield coefficients
• Models describing chemical and biochemical reactions use stoichiometric yield
coefficients to determine how much product (or biomass) will be produced from
each unit of reactant or substrate utilized.

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 • Yield coefficients describe how efficiently a reactant is converted into a product or
biomass. The formation of lactic acid from glucose during strenuous muscle activity
can be represented as:

• The yield of lactate from glucose (YLG) is 2 moles of lactate (L) per mole of glucose
(G). The relationship between lactate formation and glucose utilization would be:

• In reality, the yield will be less than 2 mol/mol as some of the carbon will be
channeled into respiration and to other cellular activities. Yield factors generally
have to be determined by experiment and ideal stoichiometries.
Bioreactors : Different Modes
• Large scale cell cultivation is performed in specialized reaction vessels known as
bioreactors or fermentors. The term fermentation is often used to describe the
cultivation of cells in fermentors. There are three main modes of bioreactor
operation: batch, continuous and fed batch (Figure 1):

• Of the three different modes,

» Fed batch bioreactors are most commonly used to produce biological
products.

» Batch reactors are the second most commonly used.

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 » Although continuous reactors are rarely used for large scale production of
biochemicals, they are widely used in waste treatment processes.
In addition, natural ecosystems, such as the rumen and gut, soil and rivers, are
more closely represented by a continuous culture system
In batch culture mode, a bioreactor is filled with fresh medium and then inoculated.
• At the end of the fermentation,
• the contents are removed for down stream processing
• the reactor is then cleaned
• sterilized and
• refilled for the next fermentation.
In continuous cultures mode,
• fresh media is continuously added into the bioreactor and at the same time
• bioreactor fluid is continuously removed.
• The cells thus continuously propagate on the fresh medium entering the reactor
and at same time, products, metabolic waste products and cells are removed in the
effluent.
Continuous culture reactors need to be shut down less frequently than batch
systems. Cells can also be immobilized in the reactor to maximize their retention and
thus increase productivity.
• With fed batch cultures, fresh media is added to the bioreactor without continuous
removal. When the reactor volume is full, the fermenter is emptied, either partially
or completely, and the process restarted.
• From the above descriptions, it can be seen that the descriptions or "models" of our
bioreactor system will need to also consider the flow of nutrients into the
fermenter and/or the removal of cells and their products.

• The batch culture is the simplest of the 3 modes of bioreactor operation and
therefore represents an appropriate starting point for studying fermentation
kinetics.
Specific Growth Rate
• As cell numbers increase, the "bio-" reaction rate will also increase. Thus if other
conditions remain constant, then the rate of increase in cells (or biomass) is
dependent on the concentration of cells present in the reactor. This can be
described as follows:

where X = concentration of biomass in the bioreactor

• Biomass concentrations are typically expressed in g/l of Dry weight
•
• The proportionality expression in Equation 12 can be replaced with a constant,
known as the specific growth rate (µ). Equation 12 thus can be re-written as:

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 where µ = Specific Growth Rate.

• This model of microbial growth is referred to as the exponential growth model.

The Growth Limiting Nutrient
• Nutrient availability has a major influence on the specific growth rate. If a nutrient
is available in concentrations that limit the growth of the cells, then that nutrient is
termed a growth limiting nutrient. When the growth limiting nutrient is a major
carbon and energy source, then it is referred to as a growth limiting substrate.

• Some fermentation media are designed such that at the end of the fermentation,
only the carbon source is in limiting supply.
• Others are designed such that nitrogen availability is growth limiting. Examples of
such fermentations are the production of PHB and algal pigments.

• In large fermentation systems O2 availability will unavoidably be rate limiting for

much of the fermentation.

• In some fermentations, the availability nutrients other than the carbon and energy
source is intentionally controlled so as to slow or even prevent cell growth. This is
done to ensure that the cells catalyze the conversion of a substrate to a product
and not use the substrate for the synthesis of cellular building blocks
• Biomass growth is dependent on nutrient availability.

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 • As a concentration of nutrient becomes growth limiting, the specific growth rate
reduces until growth ceases due to the unavailability of that nutrient. A typical plot
of specific growth rate against the concentration of a growth limiting nutrient is
shown in figure 4.
The Maximum Specific Growth Rate (µm)
• The maximum specific growth rate (µm) is the maximum rate at which cells will
grow at.

• The maximum specific growth rate (µm) is the maximum growth rate achievable
when the concentration of the growth limiting nutrient is not limiting. The higher
the value of µ then the faster the rate at which the organism can grow.
Fermentation Yield Coefficients
• A yield (or yield coefficient) is defined as the amount of product produced from a
given input.
• For example, if 0.6 g of citric acid is produced from 1 g of glucose , then the yield
of citric acid from glucose is 0.6 g/g.
• Yields can vary considerably during a fermentation. For this reason, average yields
are often used when describing production efficiencies.
• The average yields are referred to as yield coefficients.
The importance of yield coefficients
• We have already seen that yield coefficients are measure of the efficiency of a
particular conversion. Improved yield coefficients mean higher production from less
raw materials used. They are used to compare fermentation alternatives and as we
shall see later, they are used with kinetic data to model the fermentation.

• A forward looking company will put a considerable effort into improving yields.

• Higher yields will mean more product for a lower substrate input.
Types of yield coefficients
• There are two types of yields which biotechnologists are most commonly interested
in:
Biomass Yield Coefficient (Yxs)

• Biomass yields (Yxs) and

• Product yields (Yps).
• The biomass yield is the average weight of biomass produced per weight of
substrate utilized. For a batch culture, Yxs is calculated as:

where
X0 = initial concentration of biomass
S0 = initial concentration of substrate
X1 and S1 are the respective biomass and substrate concentration after a given
time (typically towards the end of the fermentation).
Product Yield Coefficient (Yps)
• Product yield coefficient is similarly calculated as:

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 where
S0 and S1 are the initial and final substrate concentrations
P0 and P1 are the initial and final product concentrations

• Sometimes product yields are represented with respect to the biomass production

• The latter yield expressions are sometimes used when quantifying the production of
secondary metabolites.

Fermentation Kinetics : Summary

• Finally we can describe microbial growth in terms of three equations:

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