Fermenter Design

PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila

College of Engineering and Technology
Department of Chemical Engineering
CHE 512:
FERMENTATION
TECHNOLOGY
Submitted by:
REBALDE, JONIEL A.
VIRATA, MA. PATRICIA S.
Submitted to:
Dr. DENVERT C. PANGAYAO
October 9, 2019
ChE 512 | Fermentation Technology | Rebalde • Virata 1
TABLE OF CONTENTS
FERMENTATION .............................................................................................................. 3
FERMENTATION TECHNOLOGY ..................................................................................... 3
MODES OF OPERATION ................................................................................................. 4
NUTRITIONAL REQUIREMENTS OF MICROORGANISMS IN FERMENTATION ............. 7
KINDS OF FERMENTATION TECHNOLOGY ................................................................. 11
FERMENTATION MEDIA ................................................................................................ 14
CHEMICAL PROCESS OF FERMENTATION ................................................................. 18
FERMENTER DESIGN AND CELL KINETICS .................................................................... 23
INTRODUCTION ............................................................................................................. 23
CELL KINETICS ............................................................................................................... 25
GROWTH CELL FOR BATCH CULTIVATION ................................................................ 26
FERMENTER DESIGN ..................................................................................................... 31
STIRRED – TANK FERMENTER ..................................................................................... 37
BATCH OR PLUG-FLOW FERMENTER ....................................................................... 45
PRODUCTIVITY IN FERMENTATION PROCESS ......................................................... 50
MULTIPLE FERMENTER CONNECTED IN SERIES ....................................................... 53
REFERENCES.................................................................................................................. 56

FERMENTATION
Fermentation is the chemical breakdown
of a substance by bacteria, yeasts, or other
microorganisms. It is a metabolic process that
produces chemical changes in organic
substrates through the action of enzymes. It is
narrowly defined as the extraction of energy
from carbohydrates in the absence of oxygen.
According to Tortora, the different definitions of fermentation are the following:
1. Preservation methods for food via microorganisms.
2. Any process that produces alcoholic beverages or acidic dairy products.
3. Any large-scale microbial process occurring with or without air.
4. Any energy-releasing metabolic process that takes place only under
anaerobic conditions.
5. Any metabolic process that releases energy from a sugar or other organic
molecule, does not require oxygen or an electron transport system, and
uses an organic molecule as the final electron acceptor.
The science of fermentation is known as zymology. The word "ferment" is
derived from the Latin verb fervere, which means to boil.
FERMENTATION TECHNOLOGY
Fermentation technology is a field which
involves the use of microorganisms and enzymes
for production of compounds which have
application in the energy, material,
pharmaceutical, chemical and the food industry.
Fermentation technology is the use of organisms to

produce food, pharmaceuticals and alcoholic beverages on a large-scale

industrial basis.
The basic principle involved in the industrial fermentation technology is that
organisms are grown under suitable conditions, by providing raw materials
meeting all the necessary requirements such as carbon, nitrogen, salts, trace
elements and vitamins. The end products formed as a result of their metab-olism
during their life span are released into the media, which are extracted for use by
human being and that have a high commercial value. The major products of
fermentation technology produced econom-ically on a large-scale industrial
basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics,
complete proteins, enzymes and other useful products.
MODES OF OPERATION
 Batch Operation
This term is attributed to that type of fermentation wherein there is
change in culture medium, number of microorganisms and the amount of the
product produced. Fermentation is carried out in a closed fermenter, with
nothing added or removed during the process. Microorganisms and nutrients
are left for a set period of time, during which the nutrient stock is depleted. It
is the most common mode of operation.
Batch fermentation
goes through a series of
phases. The microbial growth
curve can be applied. There
is a lag phase in which cells
adjust to their environment;
then a phase in which exponential growth occurs. Once many of the nutrients
have been consumed, the growth slows and becomes non-exponential, but
production of secondary metabolites (including commercially important

antibiotics and enzymes) accelerates. This continues through a stationary

phase after most of the nutrients have been consumed, and then the cells
die. Primary metabolites include alcohols such as ethanol, lactic acid, and
certain amino acids
Secondary metabolites are typically organic compounds produced

through the modification of primary metabolite synthases. Secondary
metabolites do not play a role in growth, development, and reproduction like
primary metabolites do, and are typically formed during the end or near the
stationary phase of growth.
 Fed-Batch Operation
In this type of fermentation, freshly prepared culture media is added at
regular intervals without removing the culture
fluid. This increases the volume of the
fermentation culture. This type of fermentation
is used for production of proteins from
recombinant microorganisms. It uses a semi-
closed system of cultivation and it is a variation
of batch operation. Nutrients are added slowly
and continuously during the course of fermentation. Gives higher yield

compared to batch operation since it satisfies the nutrients needed as time

passes by.
 Continuous Operation
In this type of
fermentation, the products
are removed continuously
along with the cells and the
same is replenished with the
cell girth and addition of
fresh culture media. This results in a steady or constant volume of the contents
of the fermenter. This type of fermentation is used for the production of single
cell protein (S.S.P), antibiotics and organic solvents. It is an open system of
cultivation. Involves continuous addition of fresh media and withdrawal of
fermentation product. Bacteria grows continuously in the log phase, Volume
and density remain the same in the vessel. At the log phase continuously,
which yields a steady state growth rate. Also, it can prolong the exponential
growth phase and avoid byproducts that inhibit the reactions by continuously
removing them. There are two types of continuous culture: Chemostat and
Turbidostat.
 Chemostat Bioreactor
A chemostat is a bioreactor to which fresh
medium is continuously added, while culture liquid
containing left over nutrients, metabolic end
products and microorganisms are continuously
removed at the same rate to keep the culture
volume constant. By changing the rate with which
medium is added to the bioreactor the specific
growth rate of the microorganism can be easily controlled within limits.

 Turbidostat Bioreactor
Turbidity in Microbiology is a measure of the degree to which the medium
loses its transparency due to the cells present. A turbidostat is a continuous
microbiological culture device has feedback between the turbidity of the
culture vessel and the dilution rate. The theoretical relationship between
growth in a chemostat and growth in a turbidostat is somewhat complex, in
part because they are similar. A chemostat has a fixed volume and flow rate,
and thus a fixed dilution rate. A turbidostat dynamically adjusts the flow rate
and therefore the dilution rate to make the turbidity constant. It uses
photoelectric device to measure absorbance of the culture in the vessel.
Photocells measure the
light transmitted through
the turbid culture; when
turbidity (that is, transmitted
light detected) reaches a
certain level, a medium
pump is switched on to
return the turbidity to the
required level.
NUTRITIONAL REQUIREMENTS OF MICROORGANISMS IN FERMENTATION

The fermentation medium must meet the nutritional requirements of the
microorganism (Frost and Moss, 1987). It basically contains sources of carbon,
nitrogen, minerals, and some growth factors, such as essential amino acids and
vitamins (Volesky and Luong, 1985). Besides a source of energy, organisms require
a source of materials for biosynthesis of cellular matter and products in cell
operation, maintenance and reproduction. Some microorganisms utilize
elements in the form of simple compounds, others require more complex

compounds, usually related to the form in which they ultimately will be

incorporated in the cellular material.
 Carbon Source
Biomass is typically 50% carbon on a dry weight basis, an indication
of how important it is. Since organic substances are at the same general
oxidation level as organic cell constituents, they do not have to undergo a
primary reduction to serve as sources of cell carbon. They also serve as an
energy source. It is the most vital to organic processes.
Carbon enters the pathways of energy-yielding metabolism and is

eventually secreted from the cell as CO2 (inorganic). In fermentations, the
carbon source on a unit of weight basis may be the least expensive raw
material.
Carbohydrates have a central role in biological energetics, the
production of ATP. The progressive breakdown of polysaccharides and
disaccharides to simpler sugars is a major source of energy-rich
compounds. Carbohydrates are excellent sources of carbon, oxygen,
hydrogen, and metabolic energy. They are frequently present in the media
in concentrations higher than other nutrients and are generally used in the
range of 0.2-25%.
 Nitrogen Source
Following the carbon source, the nitrogen source is generally the next
most plentiful substance in the fermentation media. A few organisms can
also use the nitrogen source as the energy source. Most photosynthetic
organisms assimilate this element in the oxidized inorganic state, as nitrates;

its biosynthetic utilization thus involves a preliminary reduction. Many
nonphotosynthetic bacteria and fungi can also meet the needs for
nitrogen from nitrates. Some microorganisms are unable to bring about a
reduction of one or both of these anions and must be supplied with the
elements in the reduced form. In these cases, nitrogen may be supplied as
ammonia salts. Several prokaryotic groups can also utilize the most
abundant natural nitrogen source, N2, which is unavailable to eukaryotes.
This process of nitrogen assimilation is termed nitrogen fixation and involves
a preliminary reduction of N2 to ammonia. Bacteria that obtain nitrogen
directly from the atmosphere are called nitrogen-fixing bacteria, examples
are Rhizobium and Azotobacter species
Nitrogen is used for the anabolic synthesis of nitrogen-containing cellular

substances, such as amino acids, purines, DNA, and RNA. Organic sources
of nitrogen in synthetic media are specific amino acids, purines,
pyrimidines, and urea.
 Oxygen Source

It is utilized by electron transport system

as a final electron acceptor. For aerobic
bacteria, oxygen source is needed to
process cellular respiration. It is needed to
produce ATP from nutrients. Other
microorganisms are facultative, meaning it can grow with or without oxygen
source.
 Phosphorus Source
It is used for nucleic acid synthesis and construction of phospholipids.
 Vitamins
Vitamins are growth factors that fulfill specific catalytic needs in biosynthesis
and are required in only small amounts. They are organic compounds that
function as coenzymes or parts of coenzymes to catalyze many reactions. The
vitamins most frequently required are thiamin and biotin.
FACTORS FOR MEDIA DEVELOPMENT:

1. The nutrient requirements of the selected microorganism
2. The composition of available industrial nutrients
3. The nutrient properties in relation to storage and handling, pasteurization or
sterilization, processing and product purification
4. Cost of the ingredients

KINDS OF FERMENTATION TECHNOLOGY

 Microbial Biomass Production
Microbial cells (biomass) are grown commercially as continuous culture on
a large scale (1500/m3). The microbial cells including algae, bacteria, yeasts,
moulds and mush-rooms are dried
and used as a good source of a
complete protein called ‘single cell
protein (SCP)’ which serves as
human food or animal feed. Single-
cell proteins (SCP) or microbial
proteins refer to edible unicellular
microorganisms. Single-cell proteins
develop when microbes ferment
waste materials.
 Microbial Metabolites Production
During the metabolism of microbial cells, a number of compounds are
produced and many are secreted out of the cell, which can be easily
extracted and are very useful to man and animals. Therefore, fermentation
by microbial cells is carried out on an industrial scale, in order to get various
metabolites.
The metabolites produced by the microbes can be grouped into two
categories:
(a) Primary metabolites
(b) Secondary metabolites
(a) Primary metabolites:
Metabolites which are produced by the metabolism required for the
main-tenance of the minimum life process of a microbe are known as primary
metabolites. The primary metabolites are produced in abundance at an early

stage of growth. Examples of primary

metabolites are ethanol, citric, acid, glutamic
acid, lactic acid, acetic acid, acetone,
formic acid, butanol, propionic acid,
dihydroxy-acetone, glycerol etc. These
metabolites are produced by fermentation
technology applying different microbes
under varying conditions of fermentation.
(b) Secondary metabolites

Secondary metabolites are
those metabolites, which are not
produced directly by the metabolism
required for the vital life process of
microbes, instead are produced by
some specialized metabolic process.
However, most of the secondary
metabolites are derived from the
primary metabolites. The secondary metabolites include the antibiotics, alkaloids,
toxic pigments, vitamins etc. These are produced during the stationary phase of
microorganism growth.
 Microbial Enzymes Production

When microbes are cultured, they secrete some enzymes into the media
and these enzymes are extracted and widely used in several industries like
detergent, food processing, brewing and pharmaceutical. Of primary interest
among the intracellular components are microbial enzymes: catalase,
amylase, protease, pectinase, cellulase, hemicellulase, lipase, lactase,

streptokinase and many others. The largest difference between this process
and the others is that the cells must be ruptured (lysed) at the end of
fermentation, and the environment must be manipulated to maximize the
amount of the product. Furthermore, the product (typically a protein) must be
separated from all of the other cellular proteins in the lysate to be purified.
 Substrate Transformation
The fermenting microbes have got the capacity to convert an added
substrate into some more valuable product. Organisms convert a substance
found in the medium to a chemically modified form that has high commercial
level It is the process in which the transformed substrate is itself the product.
Examples are conversion of ethanol to acetic acid (vinegar), isopropanol to
acetone, glucose to gluconic acid, sorbitol to sorbose, sterols to steroids.

FERMENTATION MEDIA
In a fermentation process, the choice of the most optimum micro-
organisms and fermentation media is very important for high yield of product. The
quality of fermentation media is important as it provides nutrients and energy for
growth of micro-organisms. This medium provides substrate for product synthesis
in a fermenter.
Fermentation media consists of major and minor components.
 Major components include Carbon and Nitrogen source.
 Minor components include inorganic salts, vitamins, growth factors, anti-
foaming agents, buffers, dissolved oxygen, other dissolved gases, growth
inhibitors and enzymes.
Nutrients required for fermentation media also depend upon the type of
fermentation organisms as well as the type of fermentation process to be used.
Poor choice of fermentation media might result in poor yield of output. Types of
nutrients present in the fermentation media always determine the yield of the
product.
There are two uses of fermentation media:
 Growth media
 Fermentation media
Growth medium contains low amounts of nutrients. It is useful in creating raw

material for further fermentation processes. Fermentation media contains high
amounts of nutrients. It is used in creating final products using fermentation.
There are two types of fermentation media used in industries.

 Synthetic media
 Crude media

 Synthetic Media
Synthetic media is useful in the field
of research as each and every
component is chemically known and
the exact composition of nutrients is
predetermined. In the case of
synthetic media, variation in levels and
concentration of nutrients can be controlled. Through experimentation with
synthetic media, the effect of nutrients on growth and yield of product can
be analyzed. Product recovery is easier because synthetic media contains
pure components.
A major disadvantage is the cost of media. The most important aspect of
fermentation is that it should be economic and profitable. Synthetic media is
never used on industrial scale because it is expensive. This process in only
suitable for experimentation in a lab on a small scale. The table below is an
example synthetic medium with the indicated mass values of components.

 Crude Media
Crude media is generally used on an
industrial scale for fermentation process. Crude
media contains a rough composition of media
required for fermentation. It gives high yield of
product and contains undefined sources of
ingredients. Crude media contains high level of
nutrients, vitamins, proteins, growth factors,
anti-foaming agents and precursors. It is
important to ensure that crude media should not contain toxic substances
that could affect the growth of bacteria and yield of product.
Ingredients of Crude Media:

1) Inorganic nutrients
Crude media contains inorganic salts containing cations and anion
along with a carbon source. Sometimes, fermentation micro-organisms
have a specific requirement of ions like magnesium ions, phosphates or
sulphates. These requirements are fulfilled by addition of these ions to
balance the crude media.
2) Carbon source
Simple to complex carbohydrates can be added to media as a
source of carbon. We can add different sugars like mannitol, sorbitol,
organic acids, fatty acids, proteins, peptides we can choose any of these
as a source of carbon. The selection of carbon source depends upon the
availability as well as the cost of raw material. In most of the fermentation
media, crude source of carbon is added.
 Simple carbohydrates – simple sugars are semi purified
polysaccharides and sugar alcohol are added. Sources of

simple carbohydrates are Black strap molasses, Corn molasses,

Beet molasses, sulphite waste liquor, Hydrol (corn sugar
molasses), Cannery waste.
 Complex carbohydrates – Source of complex carbohydrates
are Starch, Corn, Rice, Rye, Milo, wheat potatoes etc. Source
of starch cellulose are corn cobs, straws, wood waste, saw
meal etc.
3) Nitrogen source
Salts of urea, ammonia, and nitrate can be used as a nitrogen source.
When fermentation organisms are non-proteolytic in nature, pure form of urea,
ammonia and nitrate are used as a source of nitrogen. When fermentation
organisms are proteolytic in nature, animal and plant raw material is used; like
distillery dried solubles, Casein, Cereal grains, peptones, yeast extract,
hydrolysate, and soybean meal etc.
4) Growth factors
Crude media constituents provide enough amount of growth factors so no
extra addition of growth factor is required. If there is a lack of any kind if vitamins
or nutrients, growth factors can be added to media. Examples are yeast extract,
and beef extract.
5) Precursors
Precursors are generally present in the media as crude constituents.
Precursors are added in the fermentation media at time of fermentation as it gets
incorporated in the molecules of product without bringing any kind of change to
the final product. This helps in improving yield and quality of product. Sometimes,
precursors are added in pure form depending upon the need of product. For
example, Cobalt chloride is added less than 10 ppm in fermentation of vitamin
B12.
6) Buffers
Buffers are used to control drastic changes of pH. Sometimes, media
components may act as buffers. For example, protein, peptides, mino-acids act
as good buffers at neutral pH. Sometimes inorganic buffers like K2HPO4, KH2PO4,
and CaCO3 etc, can be added as required. Generally, during the fermentation
process, pH changes to acidic or alkaline pH. The cheapest and easily available
buffer is CaCO3.
CHEMICAL PROCESS OF FERMENTATION

The process of fermentation starts with glycolysis. Glycolysis can occur with
or without oxygen. In the presence of oxygen, glycolysis is the first stage of cellular
respiration. In the absence of oxygen, glycolysis allows cells to make small
amounts of ATP through the process of fermentation. Glycolysis takes place in the
cytosol of the cell's cytoplasm.
Glycolysis is divided into two parts: Energy-Requiring Phase and Energy-
Releasing Phase. The starting molecule of glucose gets rearranged, and two
phosphate groups are attached to it. The phosphate groups make the modified
sugar—now called fructose-1,6-bisphosphate—unstable, allowing it to split in half
and form two phosphate-bearing three-carbon sugars. Because the phosphates
used in these steps come from ATP, two ATP molecules get used up. In the second
half of glycolysis, the three-carbon sugars formed in the first half of the process go
through a series of additional transformations, ultimately turning into pyruvate. In
the process, four ATP molecules are produced, along with two molecules of
NADH.

Step 1. A phosphate group is transferred from ATP to glucose, making glucose-6-

phosphate. Glucose-6-phosphate is more reactive than glucose, and the
addition of the phosphate also traps glucose inside the cell since glucose with a
phosphate can’t readily cross the membrane.
Step 2. Glucose-6-phosphate is converted into its isomer, fructose-6-phosphate.
Step 3. A phosphate group is transferred from ATP to fructose-6-phosphate,

producing fructose-1,6-bisphosphate. This step is catalyzed by the enzyme
phosphofructokinase, which can be regulated to speed up or slow down the
glycolysis pathway.
Step 4. Fructose-1,6-bisphosphate splits to form two three-carbon sugars:

dihydroxyacetone phosphate DHAP glyceraldehyde-3-phosphate. They are
isomers of each other, but only one—glyceraldehyde-3-phosphate—can directly
continue through the next steps of glycolysis.
Step 5. DHAP is converted into glyceraldehyde-3-phosphate. The two molecules

exist in equilibrium, but the equilibrium is “pulled” strongly downward, in the
scheme of the diagram above, as glyceraldehyde-3-phosphate is used up. Thus,
all of the DHAP is eventually converted.

Step 6. Two half reactions occur simultaneously: 1) Glyceraldehyde-3-phosphate,

one of the three-carbon sugars formed in the initial phase is oxidized, and 2) NAD+
is reduced to NADH. The overall reaction is exergonic, releasing energy that is
then used to phosphorylate the molecule, forming 1,3-bisphosphoglycerate.
Step 7. 1,3-bisphosphoglycerate donates one of its phosphate groups to ADP

making a molecule of ATP and turning into 3-phosphoglycerate in the process.
Step 8. 3-phosphoglycerate is converted into its isomer, 2-phosphoglycerate.
Step 9. 2-phosphoglycerate loses a molecule of water, becoming

phosphoenolpyruvate (PEP) is an unstable molecule, poised to lose its phosphate
group in the final step of glycolysis.
Step 10. PEP readily donates its phosphate group to ADP, making a second
molecule of ATP. As it loses its phosphate, PEP is converted to pyruvate, the end
product of glycolysis.

When oxygen is absent, cells may use other, simpler pathways to

regenerate NAD+. In these pathways, NADH donates its electrons to an acceptor
molecule in a reaction that doesn’t make ATP but does regenerate NAD+ so
glycolysis can continue. The main function of fermentation is to convert NADH
back into the coenzyme NAD+ so that it can be used again for glycolysis. During
fermentation, an organic electron acceptor (such as pyruvate or acetaldehyde)
reacts with NADH to form NAD+, generating products such as carbon dioxide and
ethanol (ethanol fermentation) or lactate (lactic acid fermentation) in the
process.

 Ethanol Fermentation (Alcoholic Fermentation)

Alcoholic fermentation is the
best known of the fermentation
processes, and is involved in several
important transformation, stabilization,
and conservation processes for sugar-
rich substrates, such as fruit, and fruit
and vegetable juices. In ethanol
fermentation, the pyruvate produced
through glycolysis is converted to ethanol and carbon dioxide in two steps.
First, the pyruvate releases carbon dioxide to form a two-carbon compound
called acetaldehyde. Next, acetaldehyde is reduced by NADH to ethanol,
thereby regenerating the NAD+ for use in glycolysis. Overall, one molecule of
glucose is converted into two molecules of carbon dioxide and two molecules
of ethanol. Ethanol fermentation is typically performed by yeast, which is a
unicellular fungus.
 Lactic Acid Fermentation
It is a metabolic process by
which glucose and other six-
carbon sugars are converted into
cellular energy and the
metabolite lactate. The pyruvate
product of glycolysis gets further
acted upon under anaerobic
conditions by the enzyme lactate
dehydrogenase (LDH). In this reaction, the hydrogen from the NADH molecule
is transferred to the pyruvate molecule. This results in its carbon-oxygen double
bond being reduced to a carbon-oxygen single-bond

There are two main types of lactic acid fermentation: homolactic and
heterolactic. In homolactic acid fermentation, NADH reduces pyruvate
directly to form lactate. This process does not release gas. Overall, one
molecule of glucose is converted into two molecules of lactate. In
heterolactic fermentation, some lactate is further metabolized, resulting in
ethanol and carbon dioxide via the phosphoketolase pathway.
FERMENTER DESIGN AND CELL KINETICS
INTRODUCTION
Understanding the growth kinetics of microbial, animal, or plant cells is
important for the design and operation of fermentation systems employing them.
Cell kinetics deals with the rate of cell growth and how it is affected by various
chemical and physical conditions
Cell kinetics is the result of numerous complicated networks of biochemical

and chemical reactions and transport phenomena, which involves multiple
phases and multicomponent systems. During the course of growth, the
heterogeneous mixture of young and old cells is continuously changing and
adapting itself in the media environment which is also continuously changing in

physical and chemical conditions. As a result, accurate mathematical modeling

of growth kinetics is impossible to achieve. Even with such a realistic model, this
approach is usually useless because the model may contain many parameters
which are impossible to determine.
Therefore, we must make assumptions to be able to arrive at simple models
which are useful for fermenter design and performance predictions. Various
models can be developed based on the assumptions concerning cell
'components and population as shown in Table 6.1 (Tsuchiya et al., 1966). The
simplest model is the unstructured, distributed rnodel which is based on the
following two assumptions:
1. Cells can be represented by a single component, such as cell mass, cell
number, or the concentration of protein, DNA, or RNA. This is true for
balanced growth, since a doubling of cell mass for balanced growth is
accompanied by a doubling of all other measurable properties of the cell
population.
2. The population of cellular mass is distributed uniformly throughout the
culture. The cell suspension can be regarded as a homogeneous solution.
The heterogeneous nature of cells can be ignored. The cell concentration
can be expressed as dry weight per unit volume.
Table 6.1 Various Model for cell Kinetics
Population Cell Component
Unstructured Structured
Distributed Cells are represented by Multiple cell
a single component components, unformly
which is uniformly distributed throughout
distributed throughout the culture interact with
the culture each other

Segregated Cells are represented by Cells are composed of

a single component, but multiple components
they form a and form a
heterogeneous mixture heterogeneous mixture
the assumptions for the cells, the medium is formulated so that only one
component may be limiting the reaction rate. All other components are present
at sufficiently high concentrations, so that minor changes do not significantly
affect the reaction rate. Fermenters are also controlled so that environmental
parameters such as pH, temperature, and dissolved oxygen concentration are
maintained at a constant level.
CELL KINETICS
The growth rate based on the number of cells and that based on cell
weight are not necessarily the same because the average size of the cells may
vary considerably from one phase to another. When the mass of an individual cell
increases without division, the growth rate based on cell weight increases, while
that based on the number of cells stays the same. However, during the
exponential growth period, which is the phase that we are most interested in from
an engineer's point of view, the growth rate based on the cell number and that
based on cell weight can be assumed to be proportional to each other.
Sometimes, the growth rate can be confused with the division rate, which
is defined as the rate of cell division per unit time. If all of the cells in a vessel at
time t =0 (CN =CN) have divided once after a certain period of time, the cell
populAftion will have increased to CN x 2. If cells are divided n times after the time
t, the total number of cells will be
𝐶𝑁 = 𝐶𝑁0 ∙ 2𝑁
CN = Cell number density, number of cells per unit volume

And the average division rate is

𝑁
𝛿=
𝑡
Thus
log 2 𝐶𝑁 ∙ log 2 𝐶𝑁0
𝛿=
𝑡
and the division rate at time t is
𝑑 log 2 𝐶𝑁
𝛿=
𝑑𝑡
GROWTH CELL FOR BATCH CULTIVATION
If you inoculate unicellular microorganisms into a fresh sterilized medium and

measure the cell number density with respect to time and plot it, you may find
that there are six phases of growth and death, they are:
1. Lag phase: A period of time when the change of cell number is zero.
2. Accelerated growth phase: The cell number starts to increase and the division
rate increases to reach a maximum.
3. Exponential growth phase: The cell number increases exponentially as the cells
start to divide. The growth rate is increasing during this phase, but the division rate
which is proportional to dlnCN1 / dt, is constant at its maximum value, as illustrated
in Figure 6.1.
4. Decelerated growth phase: After the growth rate reaches a maximum, itis
followed by the deceleration of both growthrate and the division rate.
5. Stationary phase: The cell population will reach a maximum value and will not
increase any further.
6. Death phase: After nutrients available for the cells are depleted, cells will start
to die and the number of viable cells will decrease.

Exponential growth phase

In unicellular organisms, the progressive doubling of cell number results in a
continually increasing rate of growth in the population. A bacterial culture
undergoing balanced growth mimics a first-order autocatalytic chemical
reaction (Carberry, 1976; Levenspiel, 1972). Therefore, the rate of the cell
population increase at any particular time is proportional to the number density
(CN) of bacteria present at that time:
𝐶𝑁
𝑟𝑁 = = 𝜇 𝐶𝑁
𝑑𝑡
where the constant μ is known as the specific growth rate [hr-1]. The specific
growth rate should not be confused with the growth rate, which has different units
and meaning. The growth rate is the change of the cell number density with time,
while the specific growth rate is

1 𝑑𝐶𝑁 𝑑 ln 𝐶𝑁
𝜇= ( )=
𝐶𝑁 𝑑𝑡 𝑑𝑡
Which is the change of the natural log of the cell number density with time
𝑑 ln 𝐶𝑁 𝑑 log 𝐶𝑁
𝜇= = ln 2 ( ) = 𝛿𝑙𝑛2
𝑑𝑡 𝑑𝑡
Therefore, the specific growth rate μ is equal to In2 times of the division rate, δ.
If μ is constant with time during the exponential growth period, it can be
integrated from time t1 to t as
𝐶𝑁 𝑡
𝑑𝐶𝑁
∫ = ∫ 𝜇𝑑𝑡
𝐶𝑁0 𝐶𝑁 𝑡0
to give
𝐶𝑁 = 𝐶𝑁0 𝑒 [𝜇(𝑡−𝑡0 )]
Where CN is the cell number concentration at t when the exponential
growth starts. The equation shows the increase of the number of cells
exponentially with respect to time.
time required to double the population, called the doubling time (td), or
sometimes called generation time, can be estimated from by setting CN = 2CN0
and t = 0 and solving for t:
ln 2 1
𝑡(𝑑) = =
𝜇 𝛿
Example: What is the generation time of a bacterial population that increases

from 10,000 cells to 10,000,000 cells in four hours of growth?
107 4
𝑑𝐶𝑁
∫ = ∫ 𝜇𝑑𝑡
104 𝐶𝑁 0
μ=1.7269 hr-1
ln 2
𝑡(𝑑) = = 0.4014 ℎ𝑟
1.7269

Bacterial growth rates during the phase of exponential growth, under standard
nutritional conditions (culture medium, temperature, pH, etc.), define the
bacterium's generation time. Generation times for bacteria vary from about 12
minutes to 24 hours or more. Generation times for a few bacteria are shown in the
table below:
Generation time for some common bacteria under optimal conditions of growth
Bacterium Medium Generation Time
(minutes)
E choli Glucose Salt 17
Bacillus megaterium Sucrose Salt 25
Streptococcus lactis Milk 26
Streptococcus lactis Lactose broth 48
Staphylococcus aureus Heat infusion broth 27-30
Lactobacillus Milk 66-87
acidophilus
Rhizobium japonicum Mannitol salts yeast 344-461
extract
Treponema pallidum Rabbit testes 1980
FACTORS AFFECTING SPECIFIC GROWTH RATE

Substrate Concentration: One of the most widely employed expressions for
the effect of substrate concentration on μ is the Monod equation, which is an
empirical expression based on the form of equation normally associated with
enzyme kinetics or gas adsorption

where Cs is the concentration of the limiting substrate in the medium and

Ks is a system coefficient.
According to the Monod equation, further increase in the nutrient
concentration after J.1 μ reaches μ max does not affect the specific growth rate,
as shown in Figure 6.2. However, it has been observed that the specific growth
rate decreases as the substrate concentration is increased beyond a certain
level.
Dependence of the specific growth rate on the concentration of the growth

limiting nutrient μ max = 0.935 hr-1, Ks =0.22 x 1 (T-4 mol/L)
Product Concentration: As cells grow they produce metabolic byproducts

which can accumulate in the medium. The growth of microorganisms is usually
inhibited by these products, whose effect can be added to the Monod equation
as follows:

The preceding equations both describe the product inhibition fairly well.
The term CPm is the maximum product concentration above which cells cannot
grow due to product inhibition.
Other Condition: The specific growth rate of microorganisms is also affected
by medium pH, temperature, and oxygen supply. The optimum pH and
temperature differ from one microorganism to another.
Stationary phase and death phase

The growth of microbial populations is normally limited either by the
exhaustion of available nutrients or by the accumulation of toxic products of
metabolism. As a consequence, the rate of growth declines and growth
eventually stops. At this point a culture is said to be in the stationary phase. The
transition between the exponential phase and the stationary phase involves a
period of unbalanced growth during which the various cellular components are
synthesized at unequal rates. Consequently, cells in the stationary phase have a
chemical composition different from that of cells in the exponential phase.
The stationary phase is usually followed by a death phase in which the
organisms in the population die. Death occurs either because of the depletion of
the cellular reserves of energy, or the accumulation of toxic products. Like growth,
death is an exponential function. In some cases, the organisms not only die but
also disintegrate, a process called lysis.
FERMENTER DESIGN
Fermenter is basically a device in which the organism are cultivated to form the
desired product. It is a containment system designed to give right metabolic
activity of the organism

Batch fermenters are usually stirred tanks with jackets and/or coils for heating and
cooling and spargers or other means for introducing air. A typical design is shown
in the figure below.
Good mixing is important in fermentation, to ensure that all the

microorganisms in the fermenter have access to the desired concentrations of
substrates and oxygen and to maintain isothermal conditions. Baffles are usually
used to improve the mixing pattern in the vessel and prevent swirl, but baffles can
make cleaning and sterilization more difficult.
Oxygen mass transfer that is needed, and the agitation rate can be
adjusted to give the desired mass transfer parameter, kLa. The cells have a density
that is very close to the density of water, so they are easily suspended in solution
and biomass suspension rarely limits the agitation rate. Very high agitation rates
are avoided, as high shear can cause breakage of the cell walls, causing death.
Foaming can be a serious problem in fermentation. Surfactants may be
present in the growth media or formed during fermentation. The bubbling of air

naturally causes froth to form at the vapor-liquid interface. If foaming is excessive,

cells and product can be lost to the vapor recovery system and reactor
productivity impaired. Mechanical foam breakers mounted on the agitator shaft
can be used to break up large bubbles. Antifoaming agents (antifoam) can be
added if a suitable compound can be identified that does not interfere with the
cells or impede oxygen transfer. The reactor is usually designed to operate less
than 75% filled, to allow space for foam breaking and vapor-liquid segregation.
The rate of heat release in fermentation processes is usually relatively low and
adequate cooling can be provided by an external jacket or internal coils for
smaller fermenters. As was the case for baffles, the presence of coils can make
cleaning and sterilization more difficult.
The size of a batch fermenter is determined by the species productivity,
required residence time, and desired plant attainment rate. Larger size fermenters
are custom-built, but standard vessel sizes are used at small and intermediate
scale. Standard sizes are usually stated in liters or m and the more common sizes
are given in the table below with approximate equivalents in US gallons. The
vessel aspect ratio is usually between 2 and 4.
Vessel 0.5 1.0 1.5 3.0 5.0 7.5 15 25 30
size (m3)
Vessel 150 300 400 800 1500 2000 4000 7000 8000
size (gal)
When the batch residence time is long, it is common to use several reactors
in parallel, so as to maximize the productivity of downstream separation
equipment. Very large fermenters are only used for inexpensive products, so that
contamination of a batch does not cause excessive financial loss.
Most production-scale batch fermenters are made from austenitic stainless
steel, typically 316 L, to avoid contamination of the growth medium with corrosion

products. Stainless steel fermenters are designed as pressure vessels so as to

withstand sterilization conditions. At smaller production volumes, there is a
growing market for single-use disposable plastic reactors, which now account for
about 30% of the bioprocessing market. Disposable reactors are available in sizes
up to 2 m3 at time of writing.
Smaller batch fermenters are often emptied by pressuring the vessel with
air to force the liquid out through the bottom drain line. This obviates using a
bottoms pump and removes a potential source of contamination at the pump
shaft seal.
Necessary Requirements for Fermenter
a. The agitator (impeller) for mixing
Agitators achieve the following objectives: (a) bulk fluid and gas-phase mixing,
(b) air dispersion, (c) oxygen transfer, (d) heat transfer, (e) suspension of solid
particles, and (f) maintenance of a uniform environment throughout the vessel.
These objectives are achieved by a suitable combination of the most appropriate
agitator, air sparger and baffles, and the best positions for nutrient feeds, acid or
alkali for pH control and antifoam addition.
The size and position of the impeller in the vessel depends upon the size of the
fermenter. In tall vessels, more than one impeller is needed if adequate aeration
agitation is to be obtained. Ideally, the impeller should be 1/3 or 1/2 of the vessel
diameter (D) above the base of the vessel. The number of impeller may vary from
size to size to the vessel. As shown in the figure below, agitators are of several
different types.

Types of Agitators: (a) disc turbine (b) vane disc (c) open turbine, variable pitch
(d) marine propeller
b. Stirrer glands and bearings meant for aseptic sealing

The satisfactory sealing of the stirrer shaft assembly has been one of the most
difficult problems; this is very important for maintaining aseptic conditions over
long periods. Four basic types of seal assembly have been used in fermenters: (1)
the stuffing box (packed-gland seal), (2) the simple bush seal, (3) the mechanical
seal, and (4) the magnetic drive.
c. Baffles for checking the vortex resulting into foaming
The baffles are normally incorporated into agitated vessel of all sizes to prevent a
vortex and to improve aeration efficiency. They are metal strips roughly one-tenth
of the vessel diameter and attached radially to the walls.
Usually, four baffles are used, but larger fermenters may have 6 or 8 baffles. Extra
cooling coils may be attached to baffles to improve cooling. Further, the baffles
may be installed in such a way that a gap exists between the baffles and the
fermenter wall. This would lead to a scouring action around and behind the

baffles, which would minimize microbial growth on the baffles and the fermenter
wall.
d. The sparger (aeration) meant for introducing air into the liquid
A sparger may be defined as a device for introducing air into the liquid in a
fermenter. It is important to know whether sparger is to be used on its own or with
mechanical agitation as it can influence equipment design to determine initial
bubble size. Three basic types of sparger have been used and may be described
as the porous sparger, the orifice sparger and the nozzle sparger.
e. Microbial Sensor
A microbial sensor consists of a microorganism immobilized on a membrane and
an electrode. The principle of working of biosensor is the change in respiration or
the amount of metabolites produced as a result of the assimilation of substrate by
the microorganism. A wide range of thermophilic microbes have been used for
the manufacturing of microbial sensors as given in the table below.
Microorganism Detected Substance Substance to be measured

Citirobacter freundii IT Cephalosporin
Lactobacillus arabinosus H+ Vitamin-nicotinic acid
Escherichia coli CO2 L-glutamate
Streptococcus faecalis NH3 L-arginine

Trichosporon brassicae O2 Alcohol-ethanol

Clostridium butyricum Fuel Cell Formate
Yeast cells O2 Nystatin
Immobilized yeast, Trichosporon cutaneum has been used to develop an

oxygen probe for BOD estimation in sewage and other water samples. The BOD
sensor includes an oxygen electrode that consists of a platinum cathode and an
aluminum anode bathing in salt KCL solution and a Teflon membrane.
Immobilized yeast cells are crapped between the pores of porous
membrane and the Teflon sensor can measure BOD at 3-60/mg/L.
Methanotrophic bacteria namely Methylomonas Flagellata used in
measuring methane as well as oxygen according to the reaction given below:
CH4 + NADH2 + O2 CH3OH + NAD+ +H2O
Similarly, ammonia and nitrate biosensors consist of immobilized
nitrifying bacteria, Nitrosomonas europaea and a modified oxygen electrode.
This is used to determine ammonia in waste water based on the conversion of
nitrate to N2O by an immobilized denitrifying Agrobacterium sp. The nitrate
biosensor has been used to measure nitrate profiles in biofilm.
STIRRED – TANK FERMENTER
For a large-scale operatioN, the stirred-tank fermenter (STF) is the most

widely used design in industrial fermentation. It can be employed for both aerobic
and anaerobic fermentation of a wide range of cells including microbial, animal,
and plant cells.
The STF can be also used for high viscosity media. It was one of the first
largescale fermenters developed in the pharmaceutical industries. Its
performance and characteristics are extensively studied. Since a stirred-tank

fermenter is usually built with stainless steel and operated in mild operating
conditions, the life expectancy of the fermenter is also long.
Cutaway diagram of fermenter used for penicillin Production

Stirred-tank fermenters typically follow general guidelines in order to
optimize mixing and reduce power requirements. Extensive research has been
performed to give guidelines on sizing of stirred tank fermenters and their
components. However, none of these guidelines are absolute; rather they are
meant to direct the basic geometric design of stirred tank fermenters while other
factors are held constant.
a. Impeller
The ratio of the impeller diameter to the diameter of the tank (di/dt) should be
between 0.3 and 0.5. In the case of using radial flow impellers the ratio should be
approximately 0.3. If the impellers are too small they will not generate enough
fluid movement, whereas if they are too large they require much more power and
become less efficient. Typically stirred tank fermenters employ Rushton turbines
using either a single impeller or a set of impellers for tank mixing. Recent
developments in impeller design have led to the use of several different types of
impellers (e.g. Smith, He3, A320, Intermig). Even though these new types of
impellers claim to produce better mixing and have less power consumption,
typical fermenters only employ standard Rushton turbines.

b. Impeller spacing
The spacing between impellers should be 1.0di to 2.0di, where di is the diameter
of the impeller. In addition, the bottom-most impeller should be located 1.0di from
the bottom of the tank. If the impellers are spaced too close together (less than
1.0di) the power imparted to the fluid can get as low as 80% of that obtained from
proper spacing. On the other hand, if the impellers are spaced too far apart the
fluid does not experience adequate mixing. Thus, the number of impellers can be
determined from the following equation:
where HL is the height of liquid in the vessel and ni is the number of impellers
However, this is assuming all the impellers are spaced equally between the
bottom of the tank and the liquid surface. As stated before, the bottom-most
impeller is usually spaced one impeller diameter from the tank bottom, and the
upper-most impeller is spaced 1.5 or more impeller diameters from the liquid
surface
c. Baffling
Stirred tank fermenters generally use baffles because of the need to disrupt
the bulk fluid flow in the tank. Bioreactors do not need this disruption. In most
cases, four flat baffles on 90° centers are used and have a width of .08dt to .10dt,
where dt is the diameter of the tank. For low-viscosity flows baffles are attached
directly to the wall of the tank, but for moderate to high-viscosity flows baffles are
set a small distance away from the wall. While the flat, four-baffle configuration is
most common, other sizes, shapes and number of baffles have been researched,
but only on a limited basis.

d. Tank height
The height to diameter ratio of the tank is typically between 2.0 and 3.0;
however, taller tanks (up to HL/dt = 4.0) have been used to reduce the power
requirement of the impellers. Typical tanks also employ a dish-shaped bottom to
enhance mixing and prevent dead zones.
e. Stirred Power (for non-newtonian fluid)
Some fermentation broths are highly viscous, and many are non-Newtonian
liquids. For liquids with viscosities up to approximately 50 Pa s, impellers can be
used, but for more viscous liquids special types of impeller, such as the helical
ribbon-type and anchor-type, are often used.
When estimating the stirrer power requirements for non-Newtonian liquids,

correlations of the Power number versus the Reynolds number (Re) for Newtonian
liquids are very useful.
Ideal Continuous Stirred Tank Fermenter

Microbial populations can be maintained in a state of exponential growth
over a long period of time by using a system of continuous culture. Figure 6.7
shows the block diagram for a continuous stirred tank fermenter (CSTF). The
growth chamber is connected to a reservoir of sterile medium. Once growth has
been initiated, fresh medium is continuously supplied from the reservoir.

Figure 6.7 Continuous Stirred-tank Fermenter (CSTF)

Continuous culture systems can be operated as chemostat or as turbidostat.
 In a chemostat the flow rate is set at a particular value and the rate of
growth of the culture adjusts to this flow rate.
 In a turbidostat the turbidity is set at a constant level by adjusting the flow
rate.
It is easier to operate chemostat than turbidostat, because the former can
be done by setting the pump at a constant flow rate, whereas the latter requires
an optical sensing device and a controller, the turbidostat is recommended when
continuous fermentation needs to be carried out at high dilution rates near the
washout point, since it can prevent washout by regulating the flow rate in case
the cell loss through the output stream exceeds the cell growth in the fermenter.
The material balance for the microorganisms in a CSTF (Figure 6.7) can be written
as
𝑑𝐶x
FCxi − FCX + Vrx = V
𝑑𝑡
Where rx is the rate of cell growth in the fermenter and dCx/dt represents
the change of cell concentration in the fermenter with time.
For the steady-state operation of a CSTF, the change of cell concentration
with time is equal to zero (dCx/dt = 0) since the microorganisms in the vessel grow

just fast enough to replace those lost through the outlet stream, and Eq. (6.27)
becomes
𝑉 𝐶𝑥 − 𝐶𝑥𝑖
𝜏𝑚 = =
𝐹 𝑟𝑥
shows that the required residence time is equal to Cx -CXi' times l/rx, which is
equal to the area of the rectangle of width Cx- CXi and height l/rx on the l/rx versus
Cx curve.
If the input stream is sterile (Cxi=0), and the cells in a CSTF are I growing
exponentially'(rx =μCx), Eq. Becomes
1 1
𝜏𝑚 = =
𝜇 𝐷
where D is known as dilution rate and is equal to the reciprocal of the
residence time (𝜏𝑚 ). Therefore, for the steady-state CSTF with sterile feed, the
specific growth rate is equal to the dilution rate. In other words, the specific
growth rate of a microorganism can be controlled by changing the medium flow
rate. If the growth rate can be expressed by Monod equation, then

1 𝜇𝑚𝑎𝑥 𝐶𝑠
𝐷=𝜇= =
𝜏𝑚 𝐾𝑠 + 𝐶𝑠
From the equation, Cs can be calculated with a known residence time and
the Monod kinetic parameters as:
𝐾𝑠
𝐶𝑠 =
𝜏𝑚𝑎𝑥 𝜇𝑚𝑎𝑥 − 1
It should be noted, however, that the preceding expression is only valid when
𝜏𝑚𝑎𝑥 𝜇𝑚𝑎𝑥 > 1
Evaluation of Monod Kinetic Parameter

Rearranging Monod equation a linear relationship can be obtained as follow:
1 𝐾𝑆 1
= +
𝜇 𝜇𝑚𝑎𝑥 𝐶𝑠 𝜇𝑚𝑎𝑥
where 𝜇 is equal to the dilution rate (D) for a chemostat. If a certain
microorganism follows Monod kinetics, the plot of 1/ 𝜇 versus l/Cs yields the values
of 𝜇 max and Ks by reading the intercept and the slope of the straight line
It can be rearranged to give the following linear relationships, which can
be employed instead of Equation above for a better estimation of the parameters
in certain cases
𝜇
𝜇 = 𝜇𝑚𝑎𝑥 − 𝐾𝑠
𝐶𝑠
However, the limitation of this approach to determine the kinetic
parameters is in the difficulty of running a CSTF. For batch runs, we can even use
shaker flasks to make multiple runs with many different conditions at the same
time. The batch run in a stirred fermenter is not difficult to carry out, either. Since
there is no input and output connections except the air supply and the length of
a run is short, the danger of contamination of the fermenter is not serious.
Example
A chemostat study was performed with yeast. The medium flow rate was
varied and the steady-state concentration of cells and glucose in the fermenter
were measured and recorded. The inlet concentration of glucose was set at 100
g/L. The volume of the fermenter contents was 500 mL. The inlet stream was sterile
a. Find the rate equation for cell growth.

b. What should be the range of the flow rate to prevent washout of the cells?
Solution:
Let's assume that the growth rate can be expressed by Monod kinetics. If this
assumption is reasonable, the plot of 1/D versus 1/Cs will result in a straight line
according to linearize equation of Monod Equation.

a. The plot of 1/D versus 1/Cs is shown in Figure which shows a straight line with
1 𝐾𝑠
intercept𝜇 = 𝟑. 𝟖 .And slope 𝜇 = 𝟓. 𝟐
𝑚𝑎𝑥 𝑚𝑎𝑥
Therefore, 𝝁𝒎𝒂𝒙 = 0.26 hr-1, and Ks = 1.37 g/L. The rate equation of cell growth is
𝟎. 𝟐𝟔𝑪𝑺 𝑪𝑿
𝑟𝑥 =
𝟏. 𝟑𝟕 + 𝑪𝑺
b. To prevent washout of the cells, the cell concentration should be
maintained so that it will be greater than zero.
Solving the preceding equation for τm yields

𝑉 𝐾𝑆 + 𝐶𝑆𝑖
𝜏𝑚𝑎𝑥 = =
𝐹 𝐶𝑆𝑖 𝜇𝑚𝑎𝑥
0.5 ∙ 100 ∙ 0.23 𝑳
𝐹= = 𝟏. 𝟏𝟐𝟖
1.37 + 100 𝒉𝒓
BATCH OR PLUG-FLOW FERMENTER

An ideal stirred fermenter is assumed to be well mixed so that the
contents are uniform in composition at all times. Another ideal fermenter is the
plug-flow fermenter, the analysis of which is analogous to the ideal batch
fermenter.
In a tubular-flow fermenter, nutrients and microorganisms enter one end
of a cylindrical tube and the cells grow while they pass through. Since the long
tube and lack of stirring device prevents complete mixing of the fluid, the
properties of the flowing stream will vary in both longitudinal and radial direction.
However, the variation in the radial direction is small compared to that in the
longitudinal direction. The ideal tubular-flow fermenter without radial variations is
called a plug-flow fermenter (PFF).

In a plug flow reactor, nutrients (and sometimes organisms) are

introduced to the reactor continuously and move through the reactor as a
“plug”. The system may be either contained (as in a water main, oil pipeline, or
blood vessel) or open (as in a shower curtain, stream, or canyon seep).
In an ideal plug flow reactor, it is assumed that there is no mixing of the
medium along the long axis (X-axis) of the reactor although there may be lateral
mixing in the medium at any point along the long axis (i.e., the Y-axis).
If liquid medium is inoculated with a seed culture, the cell will start to
grow exponentially after the lag phase. The change of the cell concentration in
a batch fermenter is equal to the growth rate of cells in it
𝑑𝐶𝑋
= 𝑟𝑥 = 𝜇𝐶𝑥
𝑑𝑡
To derive the performance equation of a batch fermentation, we need
to integrate Equation to obtain:
𝐶𝑋 𝐶𝑋 𝑡
𝑑𝐶𝑋 𝑑𝐶𝑋
∫ =∫ = ∫ 𝑑𝑡 = 𝑡 − 𝑡0
𝐶𝑋0 𝑟𝑥 𝐶𝑋0 𝜇𝐶𝑥 𝑡0
According to Eq., the batch growth time t - to is the area under the 1/rx
versus Cx curve between CXo and Cx as shown. The solid line in Figure was
calculated with the Monod equation and the shaded area is equal to t - to. The
batch growth time is rarely estimated by this graphical method since the Cx versus
tcurve is a more straightforward way to determine it. However, the graphical
representation is useful in comparing the performance of the various fermenter
configurations, which is discussed later

A graphical representation of the batch growth time t- t1 (shaded area). The

solid line represents the Monad model with, 𝝁𝒎𝒂𝒙 = 0.935 hr-1, Ks =0.71 g/L, Yx/s =
0.6, Cxo = 1 g/L, and Cso = 10 g/L
this time just note that the curve is U shaped, which is characteristic of
auto catalytic reactions:

S + X -> X + X
The rate for an autocatalytic reaction is slow at the start because the
concentration of X is low. It increases as cells multiply and reaches a maximum
rate. As the substrate is depleted and the toxic products accumulate, the rate
decreases to a low value.
If Monod kinetics adequately represents the growth rate during the
exponential period, we can obtain
𝐶𝑋 (𝐾 𝑡
𝑆 + 𝐶𝑆 )𝑑𝐶𝑋
∫ = ∫ 𝑑𝑡
𝐶𝑋0 𝜇𝑚𝑎𝑥 𝐶𝑆 𝐶𝑥 𝑡0
It can be integrated if we know the relationship between Cs and Cx. It

has been observed frequently that the amount of cell mass produced is
proportional to the amount of a limiting substrate consumed. The growth yield
(YX/S) is defined as
∆𝐶𝑋 𝐶𝑋 − 𝐶𝑋0
𝑌𝑋/𝑆 = =
∆𝐶𝑆 −(𝐶𝑆 − 𝐶𝑆0 )
Combining the two equation:
It should be noted that even though the Monod equation has the same form as
the Michaelis-Menten equation, the rate equation is different.
Monod Equation
𝒅𝑪𝑿 𝝁𝒎𝒂𝒙 𝑪𝑺 𝑪𝑿
𝒓𝑿 = =
𝒅𝒕 𝑲𝑺 + 𝑪𝑺
Usage:
 Large scale
 Fast reactions
 Homogeneous reactions
 Heterogeneous reactions

 Continuous production
 High temperature
Advantage Disadvantage
 High conversion per unit  Undesired thermal gradients
volume may exist
 Low operating (labor) cost  Poor temperature control
 Continuous operation  Shutdown and cleaning may
 Good heat transfer be expensive
Comparison of batch and CSTF

Since the 1/rx versus Cx curve is U shaped, we can make the following conclusions
for single fermenter
1. The most productive fermenter system is a CSTF operated at the cell
concentration at which value of 1/rx is minimum, because it requires the
smallest residence time.
2. If the final cell concentration to be reached is in the stationary phase, the
batch fermenter is a better choice than the CSTF because the residence
time required for the batch is smaller than that for the CSTF.
Graphical illustration of the residence time required for the (a) CSTF and (b)
batch fermenter

PRODUCTIVITY IN FERMENTATION PROCESS
Productivity is synonymous with capacity to produce, the rate at which

a unit volume of plant can turn out product. In the figure above, the two diagonal

lines drawn tangent to the curve at the points shown represent productivities. The
lesser sloped line is the maximum productivity of the batch fermentation process.
Its point of tangency does not correspond, in this case, to the point of maximum
product Concentration. At this latter point, the productivity would be lower
because the slight increase in product concentration is achieved only by an
unduly long extension of the process cycle. The batch productivity line considers
the unproductive parts of the process such as harvesting and rebatching, in a
period called down time.
The steep sloped line is the maximum instantaneous productivity of the
process and represents the full potential of continuous operation. It corresponds
to the steepest slope of the reaction curve. Operating the process continuously
at the product concentration level corresponding to this slope eliminates
unproductive down time.
Operating a single stage continuously at the point of maximum
productivity always results in less efficient utilization of nutrient raw materials.
Where process economics warrant, second and later stage fermentation vessels
can be used to make fuller use of nutrients and arrive at higher product
concentrations in the harvest stream. Productivity is decreased because of the
additional volume. If the continuous operation is to remain most economical, this
decreased productivity must exceed or compare favorably with the operating
productivity of the batch process. The complete process economic picture must
be analyzed thoroughly.
One of the techniques to increase productivity in the fermentation
process is through cell immobilization. Immobilization of enzymes (or cells) refers
to the technique of confining/anchoring the enzymes (or cells) in or on an inert
support for their stability and functional reuse. By employing this technique,
enzymes are made more efficient and cost-effective for their industrial use. Some
workers regard immobilization as a goose with a golden egg in enzyme

technology. Immobilized enzymes retain their structural conformation necessary

for catalysis. The cells are retained inside the fermenter through:
 Attachment to a surface
 Entrapment within porous matrices
 Containment behind a barrier
 Self-aggregation
It would be interesting to derive the equations for the cell concentration
and residence time at this maximum cell productivity. The optimum cell
concentration for the maximum productivity is
∝
𝐶𝑥,𝑜𝑝𝑡 = 𝑌𝑋/𝑆 𝐶𝑆𝑖
∝ +1
In terms of substrate concentration
1
𝐶𝑠,𝑜𝑝𝑡 = 𝐶𝑆𝑖
∝ +1
Where
𝐾𝑆 + 𝐶𝑆𝑖
∝= √
𝐾𝑆
For Cs yields the optimum residence time:

∝
𝜏𝑚𝑎𝑥 =
𝜇𝑚𝑎𝑥 (∝ −1)

MULTIPLE FERMENTER CONNECTED IN SERIES
In continuous fermentation, the plant is operated such that the rate of

live cell loss (either by death or elutriation from the fermenter) matches the growth
rate of new cells. A stable population balance is thereby achieved, and with
careful control this steady state can be maintained for days, weeks, or even
months. Steady continuous operation maximizes the volumetric productivity of
the fermenter, as the fraction of time spent in draining, cleaning, filling, and
sterilizing operations is dramatically reduced.
Multiple CSTF in series
Hill and Robinson (1989) derived an equation to predict the minimum
possible total residence time to achieve any desired substrate conversion. They
found that three optimally designed CSTFs connected in series provided close to
the minimum possible residence time for any desired substrate conversion.
The schematic diagram of the multiple CSTFs connected in series is
shown. For the nth steady-state CSTF, the material balance for the microorganisms
can be written as
𝐹(𝐶𝑥𝑛−1 − 𝐶𝑥𝑛 ) + 𝑉𝑛 𝑅𝑋𝑛 = 0

𝝁𝒎𝒂𝒙 𝑪𝑺𝒏 𝑪𝑿𝒏

𝒓𝑿𝒏 =
𝑲𝑺 + 𝑪𝑺𝒏
Growth yield can be expressed as
𝐶𝑋𝑛 − 𝐶𝑋𝑛−1
𝑌𝑋 =
𝑆 𝐶𝑆𝑛−1 − 𝐶𝑆𝑛
We can calculate either dilution rate with the known cell concentration, or
vice versa.
Example:
Suppose you have a microorganism that obeys the Monad equation where μmax
= 0.7 hr-1 and Ks = 5 g/L. The cell yield (𝑌𝑋 ) is 0.65. You want to cultivate this
𝑆
microorganism in either one fermenter or two in series. The flow rate and the
substrate concentration of the inlet stream should be 500 L/hr and 85 g/L,
respectively. The substrate concentration of the outlet stream must be 5 g/L
a. If you use one CSTF, what should be the size of the fermenter? What is the
cell concentration of the outlet stream?
b. If you use two CSTFs , what sizes of the two fermenters will be most
productive? What are the concentration of cells and substrate in the outlet
stream of the first fermenter?
Solution:
𝜇𝑚𝑎𝑥 𝐶𝑠 0.75∗5
a. 𝐷 = = = 0.35
𝐾𝑠 +𝐶𝑠 5+5

500
V=F/ D =0.35 = 1,429 𝑙
Cx=Yx/s (Csi - Cs)= 0.65 (85-5) = 52 g/ L

b. For two CSTF in seies, the first fermenter must be operated at Cx,opt and Cs,opt
𝐾𝑆 +𝐶𝑆𝑖 5+85
∝= √ =∝= √ =4.2
𝐾𝑆 5
∝ 4.2
𝐶𝑥,𝑜𝑝𝑡 = 𝑌𝑋/𝑆 𝐶𝑆𝑖 = 0.65 ∗ 85 = 45 𝑔/𝑙
∝ +1 4.2 + 1
1 1
𝐶𝑠,𝑜𝑝𝑡 = 𝐶𝑆𝑖 = 85 = 1.6 𝑔/𝐿
∝ +1 4.2 + 1
∝ 4.2
𝜏𝑚𝑎𝑥 = = = 1.9 ℎ𝑟
𝜇𝑚𝑎𝑥 (∝ −1) 0.7(4.2 − 1)
V1=𝜏𝑚1 F= 1.9*500= 950 L
For the second fermenter (material balance)
Thus,
Vtotal= 1 142 L

The design of a continuous fermentation reactor is strongly dependent on

whether the product is extracellular or intracellular.
Extracellular products can be recovered from the fermentation broth
without requiring removal of the cells. The cells can therefore be contained in the
reactor loop, either by immobilization, or by using a reactor membrane circuit.
Reactor productivity will usually be optimized if the cells are in the stationary
phase of the growth cycle, with the highest stable concentration of live cells. The
rates of substrate addition, dilution (by water coming in with the substrate),
oxygen addition, carbon dioxide removal, and heat removal must all be
controlled to maintain the optimal conditions for sustaining this steady state. The
most common industrial example of continuous fermentation for an extracellular
product is the use of Saccharomyces cerevisiae to ferment sugars into alcohol in
production of ethanol for use in gasoline and in large-scale brewing of wine and
beer.
REFERENCES
Deindoerfer, F. H., & Humphrey, A. E. (1959). Design of Multistage Systems for
Simple Fermentation Processes. Industrial and Engineering Chemistry, 809-812.
Jha, N. (n.d.). Biology Discussion. Retrieved from Immobilization of Enzymes and

Cells: Methods, Effects and Applications:
http://www.biologydiscussion.com/enzymes/immobilization/immobilization-
ofenzymes-and-cells-methods-effects-and-applications/10208
Katoh, S., & Yoshida, F. (2009). Biochemical Engineering. Weinheim: Wiley-VCH.
Kenty, B.M., Li, Z.J., Lee, S.S., and Xing, Z., "Scale-Up Analysis for a CHO Cell
Culture Process in Large-Scale Bioreactors," Biotechnology and Bioengineering,
vol. 103, no. 4, 2009.
Kenty, B.M., Li, Z.J., Lee, S.S., and Xing, Z., "Scale-Up Analysis for a CHO Cell
Culture Process in Large-Scale Bioreactors," Biotechnology and Bioengineering,
vol. 103, no. 4, 2009.

Lumen. (n.d.). Retrieved from Microbial Growth:

https://courses.lumenlearning.com/trident-
boundlessmicrobiology/chapter/microbial-growth/
Montes, F.J., Galan, M.A., and Martin, M., "On the Contribution of the Scales of
Mixing to the Oxygen Transfer in Stirred Tanks," Chemical Engineering Journal,
vol. 145, 2008, pp. 232-241.
Nauman, E.B., Chemical Reactor Design, Optimization and Scale-Up, McGraw

Hill, New York, 2002.
Oldshue, J.Y., Fermentation and Biochemical Engineering Handbook, 2nd Ed,

William Andrew, 1997, pp. 181-241.

Fermenter Design

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Fermenter Design

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PAMANTASAN NG LUNGSOD NG MAYNILA

University of the City of Manila

Dr. DENVERT C. PANGAYAO

ChE 512 | Fermentation Technology | Rebalde • Virata 2

ChE 512 | Fermentation Technology | Rebalde • Virata 3

produce food, pharmaceuticals and alcoholic beverages on a large-scale

ChE 512 | Fermentation Technology | Rebalde • Virata 4

antibiotics and enzymes) accelerates. This continues through a stationary

Secondary metabolites are typically organic compounds produced

ChE 512 | Fermentation Technology | Rebalde • Virata 5

compared to batch operation since it satisfies the nutrients needed as time

ChE 512 | Fermentation Technology | Rebalde • Virata 6

NUTRITIONAL REQUIREMENTS OF MICROORGANISMS IN FERMENTATION

ChE 512 | Fermentation Technology | Rebalde • Virata 7

compounds, usually related to the form in which they ultimately will be

Carbon enters the pathways of energy-yielding metabolism and is

organisms assimilate this element in the oxidized inorganic state, as nitrates;

Nitrogen is used for the anabolic synthesis of nitrogen-containing cellular

ChE 512 | Fermentation Technology | Rebalde • Virata 9

It is utilized by electron transport system

FACTORS FOR MEDIA DEVELOPMENT:

ChE 512 | Fermentation Technology | Rebalde • Virata 10

KINDS OF FERMENTATION TECHNOLOGY

ChE 512 | Fermentation Technology | Rebalde • Virata 11

stage of growth. Examples of primary

(b) Secondary metabolites

 Microbial Enzymes Production

ChE 512 | Fermentation Technology | Rebalde • Virata 12

ChE 512 | Fermentation Technology | Rebalde • Virata 13

Growth medium contains low amounts of nutrients. It is useful in creating raw

There are two types of fermentation media used in industries.

ChE 512 | Fermentation Technology | Rebalde • Virata 14

ChE 512 | Fermentation Technology | Rebalde • Virata 15

Ingredients of Crude Media:

ChE 512 | Fermentation Technology | Rebalde • Virata 16

simple carbohydrates are Black strap molasses, Corn molasses,

CHEMICAL PROCESS OF FERMENTATION

ChE 512 | Fermentation Technology | Rebalde • Virata 18

Step 1. A phosphate group is transferred from ATP to glucose, making glucose-6-

Step 2. Glucose-6-phosphate is converted into its isomer, fructose-6-phosphate.

Step 3. A phosphate group is transferred from ATP to fructose-6-phosphate,

Step 4. Fructose-1,6-bisphosphate splits to form two three-carbon sugars:

Step 5. DHAP is converted into glyceraldehyde-3-phosphate. The two molecules

ChE 512 | Fermentation Technology | Rebalde • Virata 19

Step 6. Two half reactions occur simultaneously: 1) Glyceraldehyde-3-phosphate,

Step 7. 1,3-bisphosphoglycerate donates one of its phosphate groups to ADP

Step 8. 3-phosphoglycerate is converted into its isomer, 2-phosphoglycerate.

Step 9. 2-phosphoglycerate loses a molecule of water, becoming

ChE 512 | Fermentation Technology | Rebalde • Virata 20

When oxygen is absent, cells may use other, simpler pathways to

ChE 512 | Fermentation Technology | Rebalde • Virata 21

 Ethanol Fermentation (Alcoholic Fermentation)

ChE 512 | Fermentation Technology | Rebalde • Virata 22

FERMENTER DESIGN AND CELL KINETICS

Cell kinetics is the result of numerous complicated networks of biochemical

ChE 512 | Fermentation Technology | Rebalde • Virata 23

physical and chemical conditions. As a result, accurate mathematical modeling

ChE 512 | Fermentation Technology | Rebalde • Virata 24

Segregated Cells are represented by Cells are composed of

ChE 512 | Fermentation Technology | Rebalde • Virata 25

And the average division rate is

If you inoculate unicellular microorganisms into a fresh sterilized medium and