BIOPROCESS

ENGINEERING
PRINCIPLES
(ECP 424)
Mrs Fungura

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Course Specific Outcomes
• Understand the terminology and concepts of the bioprocess industries
• Understand the biological organisms used in bioprocesses and their
kinetics
• Apply the characteristics of biochemical kinetics in relation to biochemical
reactor design
• Develop stead-state and unsteady- state and control equation for different
types of biochemical reactors
• Analyse the characteristics of different types of bio- reactors
• Calculate oxygen transfer rate in aerobic bio-processes
• Understand the importance of sterilisation and containment in bio-
processes and applications
• Understand downstream processing techniques applied in bioprocesses
• Be proficient in basic laboratory methodology necessary for bioprocess
development

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TOPICS
1. Cells and Cell Kinetics
2. Enzymes and Enzymes Kinetics
3. Bioreactors Design and Control
4. Oxygen Transfer Processes
5. Containment, Sterilization and Sterilization Kinetics
6. Downstream Processes

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Biochemical engineering review

4
CELLS AND
CELL
KINETICS
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Topic Outline
• Definition of a cell
• Classification of cells
• Composition of cells
• Cell kinetics
• Batch growth kinetics
• Monod’s equation
• Continuous growth kinetics

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 CELLS
•All living organisms are made up of cells
•All organisms are composed of one or more cells
•Cells are the smallest living units of all living organisms
•Cell is the structural and functional unit of life
•Cells arise only by division of a previously existing cell

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Classification of cells
All cells share four key components:
• The plasma membrane is an outer covering that separates the cell’s
interior from its surrounding environment.
• Cytoplasm consists of the jelly-like cytosol inside the cell, plus the
cellular structures suspended in it.
• DNA is the genetic material of the cell.
• Ribosomes are molecular machines that synthesize proteins.
• Cells are either prokaryotic or eukaryotic cells
• These types are distinguished by structural organisation
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Prokaryotic cells
Characteristics
• simple, single-celled and lack a nucleus and membrane-bound
organelles
• all the intracellular water soluble components(proteins, DNA and
metabolites are located together in the cytoplasm enclosed by the cell
membrane, rather than in separate cellular components.
• Dimensions : 0.5-3microns they are significantly smaller than eukaryotic
are not divided up on the inside by membrane walls, but consist instead
of a single open space, therefore have a larger surface-area-to-volume
ratio, giving them a higher metabolic rate, a higher growth rate, and as a
consequence, a shorter generation time than eukaryotes
• Eg Bacteria

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Eukaryotic Cells
Characteristics
• A membrane-bound nucleus, a central cavity surrounded by
membrane that houses the cell’s genetic material.Have membrane
bound organelles
• A number of membrane-bound organelles, compartments with
specialized functions that float in the cytosol.
• Plant and animal cells are not the only types of eukaryotic cells.
Protists and fungi are two other types of eukaryotic organism

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Summary of differences between the
prokaryotic and eukaryotic cells
Prokaryotic Cells
Small cells (< 5 mm)
Always unicellular
No nucleus or any membrane-bound
Organelles
DNA is circular, without proteins
Ribosomes are small
No cytoskeleton
Cell division is by binary fission
Reproduction is always asexual
Eukaryotic cells
Larger cells (> 10 mm)
Often multicellular
Always have nucleus and other
Membrane-bound organelles
DNA is linear and associated with proteins
to form chromatin
Ribosomes are large
Always has a cytoskeleton
Cell division is by mitosis or meiosis
Reproduction is asexual or sexual
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 Parts of an Animal Cell
• Vacuole-cell storage sac for food, waste, and water
• Mitochondrion –produces energy in a cell
• Chromosomes-provides direction for cells to follow
• Nucleus-control center of a cell
• Endoplasmic reticulum-transportation system
• Cytoplasm-gel like substance found in a cell
• Cell membrane-surrounds cell material
• Golgi body -Processes and packages proteins and transports them to other parts of the cell or outside the cell.

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Parts of a Plant Cell
• Cytoplasm-gel like substance found in a cell
• Chloroplasts-a green structure in a plant
• Cell wall-a stiff covering that protects plant cells
• Nucleus-control center of the cell
• Chromosomes-provides direction for cell to follow
• Endoplasmic reticulum-transportation network
• Mitochondrion-produces energy in the cell
• Vacuole-cell storage sac for food, waste and water

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Chemical Composition of Cells
• All living organisms are made up of chemical substances
• Reactions between these substances keep the cytoplasm (and the
organism) alive. They are living processes.
• There are four general classes of macromolecules within living cells:
nucleic acids, proteins, carbohydrates, and lipids.
• These compounds, are created through polymerization of building
blocks that have molecular weights in the range of 50 to 150.

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Carbohydrates
• Carbohydrates are members of a large group of molecules that contain the elements carbon, hydrogen and
oxygen. Familiar carbohydrates are sugar and starch
• The carbohydrate glucose is the most common single sugar molecule and is known as a monosaccharide. It
contains carbon, hydrogen and oxygen in the ratio 1:2:1. It has six atoms of carbon, 12 atoms of hydrogen
and six atoms of oxygen; hence its chemical formula is C 6 H12O6 .
• The original source of all glucose molecules is photosynthesis. During this process, the inorganic molecules,
carbon dioxide and water, are broken apart using light energy from the Sun and enzymes of the chloroplast.
The atoms are rejoined to form glucose, water and oxygen.
• Glucose is a monosaccaride. If two monosaccharide molecules join together, they form a disaccharide (‘two
sugars’). Sucrose or common table sugar is an example of a disaccharide. If many monosaccharides join
together, or two or more disaccharides join together, or two or more polysaccharides join together, they
form a polysaccharide (‘many sugars’).
• Sugars move around in the tissues of the plant in the form of sucrose (a disaccharide) rather than glucose.
Being in the less reactive form of sucrose ensures that the sugar will reach other cells that need it.
• Cellulose, found in plant cell walls, is composed of many glucose molecules joined together. The different
linking patterns between the glucose molecules mean that cellulose fibres are tough, insoluble and resistant
to being crushed and bent. Hence, they are ideal as the structural components of plant cell walls. The
efficient functioning of plants is very much bound up with the properties of their tough, cellulose cell walls.
 Carbohydrates
• Carbohydrates provide the main source of energy for respiration in living
organisms
• Carbohydrates constitute a major class of naturally occurring organic compounds,
including sugars, starches, and celluloses.
• They are essential to the maintenance of plant and animal life. Carbohydrates are
classified into three major groups:monosaccharides, oligosaccharides, and
polysaccharides.
• Monosaccharides are the simplest carbohydrate units. Oligosaccharides contain
two or more of these simple mono saccharide units, and polysaccharides contain
hundreds or thousands of them.
Carbohydrates
• They are linked together to form long chains. Some starches contain over
6000 glucose molecules and hence are perfect molecules to act as
glucose storage.
• Most plants store excess starch in their roots and break it down into
glucose when they require it for respiration.
• Animals tend to store excess carbohydrate in the form of glycogen, a
polysaccharide. Glycogen is very similar to starch except for some slight
differences in the way that the polysaccharide molecule is bonded
together.
• Glycogen is stored in the liver and muscles and is converted back into
glucose as the concentration of glucose in the blood begins to drop
Proteins
• Proteins are made up of the elements carbon, hydrogen and oxygen and always
contain nitrogen. In addition, sulfur is often present, and sometimes phosphorus
and other elements.
• These elements combine to form building blocks called amino acids. Proteins make
up the structure of cells; cytoplasm, nucleus, cell membranes and enzymes
• There are over 20 different types of amino acids and they join together to form a
protein or polypeptide. Examples are glycine (Gly), alanine (Ala), valine (Val) and
cysteine (Cyst).
• It is the order and number of amino acids that make different types of protein. The
ordering of amino acids in proteins is determined by the genes in our
chromosomes.
• Membrane proteins control movement of substances into and out of organelles and
between the cell and its external environment.
• Many chemicals are unable to move directly through the plasma membrane.
Channels made of transport proteins that act like gates, facilitating movement
across the membrane.
Proteins
• Other proteins, called structural proteins, link membranes, cytoplasm and the
nucleus, allowing for communication.
• Structural proteins such as the insoluble fibrous protein keratin are found in hairs,
feathers, nails, hooves and horns.
• Another fibrous protein, collagen, is the most abundant protein in vertebrates,
making up a third of their total protein mass.
• Humans are largely held together by collagen as it is found in bones, cartilage,
tendons, ligaments, connective tissue and skin. Collagen is even found in the
cornea of the eye. Collagen fibres have a tensile strength greater than that of steel.
• Enzymes are one of the most important groups of proteins. Without enzymes the
reactions that occur in living organisms would be so slow as to hardly proceed at
all; this would be incompatible with the maintenance of life. They are present in
the cytoplasm of all cells
Lipids
• Lipids form a larger class of compounds containing fats and oils. Fats are solid at
room temperature and oils are liquid. They all contain the elements carbon,
hydrogen and oxygen and are insoluble in water.
• They are made up from glycerol and fatty acids.
• The most common type of lipid is the triglyceride. As its name (tri-) suggests it is
composed of three fatty acids and one glycerol. If the fatty acid contains only
single bonds between the carbon atoms, it is described as a saturated fat. Single
bonds tend to be difficult to break down by the cells in your body. As such they
can also withstand much higher temperatures.
• Phospholipids differ to triglycerides because one of the fatty acids is replaced by
a phosphate group. This causes one end of the phospholipid molecule to be
‘water-loving’ (hydrophilic) and the other end to be ‘water-hating’
(hydrophobic)
Nucleic Acid
• The nucleic acids are the building blocks of living organisms
• Examples are DNA which stands for deoxyribonucleic acid, RNA stands
for ribonucleic acid.
Salts and Water
• In addition to proteins, carbohydrates and lipids, the cytoplasm
contains salts and water
• Water makes up the bulk of cytoplasm
• All the chemical reactions in cytoplasm take place in solution, i.e. in
water
• Water itself takes part in many of these chemical reactions
• Salts of sodium, potassium and calcium and many others play an
important part in these reactions

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CELL GROWTH KINETICS
- Introduction
- Growth patterns and kinetics in batch culture
- growth phases
- effect of factors: oxygen supply
- heat generation
- Growth kinetics (Monod Equation)
- Growth in continuous culture (ideal chemostat)

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 Growth Kinetics
Introduction
Autocatalytic reaction: The rate of growth is directly related to cell
concentration.

substrates + cells → extracellular products + more cells

∑S + X → ∑P + nX

S: substrate concentration (g/L); X: cell mass concentration (g/L);

P: product concentration (g/L); n: increased number of biomass.

Net specific growth rate (1/time):

t: the time
1 dX
 net 
X dt
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 Growth Kinetics
Introduction
Net specific growth rate (1/time):

 net   g  k d
g : Gross specific growth rate (1/time)

kd : The rate of loss of cell mass due to cell death

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Batch Growth Kinetics
- Growth patterns and kinetics in batch culture
- growth phases
In batch culture:
- lag phase
- logarithmic or exponential growth phase
- deceleration phase
- stationary phase
- death phase

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Typical growth curve for a bacterial population
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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, nutrient-poor medium

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Typical growth curve for a bacterial population
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Typical growth curve for a bacterial population
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Batch Growth Kinetics
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.

• Growth rate is independent of nutrient concentration, as nutrients are in

excess.

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 The slope  net is constant.

Typical growth curve for a bacterial population

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 Batch Growth Kinetics
Exponential growth phase

Doubling time of cell mass: the time required to double the microbial mass:

ln X / X 0 ln 2 0.693
d   
 net  net  n et

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Typical growth curve for a bacterial population
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Batch Growth Kinetics
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
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Typical growth curve for a bacterial population
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Batch Growth Kinetics
Stationary Phase:

With the exhaustion of nutrients (S≈0) 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.
Primary metabolites are growth-related: ethanol by S. cerevisae.
Secondary metabolites are non-growth-related: antibiotics,
pigments.
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Batch Growth Kinetics
Stationary phase

- Cell lysis (spillage) may occur and viable cell mass may drop.
A second growth phase may occur and cells may grow on lysis
products of lysed cells (cryptic growth)

The rate describing the conversion of cell mass into maintenance

energy or the loss of cell mass due to cell lysis:

dX
 kd X
dt
k d is the rate constant for endogenous metabolism . 38
Batch Growth Kinetics
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
'
k d is the first - order death rate constant.
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Growth Kinetics

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Growth of cells
• Cell growth is the primary response of viable cells to substrates and
nutrients.
• Substrates/nutrients + cells → products + more cells
• Product formation is a secondary response
dX
 X
. dt
Exponential Phase Growth
1. Nutrient and substrate concentrations are large
2. Growth rate is independent of nutrient and substrate conc.
3. Cell number and mass concentrations increase exponentially

dX
  net X , X  X 0 at t  0
dt
Integration of the above equation yields:
X
ln  μnet t , or X  X 0e  n ett
X0
X and X 0 are cell concentrations at time t and t  0

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Yield Coefficient
• Yield coefficients, Y, are defined based on the amount of consumption
of another material
• This is commonly refered to as the substrate to biomass yield, and is
used to convert between cell growth rate dX/dt and substrate
utilization rate dS/dt
Yield Coefficient
• In order to model the amount of substrate and product being consumed/produced in following equations, yield
coefficients are utilized. Ysc and Ypc are the yield coefficients for substrate-to-cells and product-to-cells,
respectively.
• Yield coefficients have the units of g variable/g cells.
• rate of substrate depletion :
−𝑟𝑠=𝑌𝑠𝑐𝑟𝑔+𝑚𝐶𝑐

• rate of product formation:

𝑟𝑝=𝑌𝑝𝑐𝑟𝑔

Where
• rg = cell growth rate
• Cc = cell concentration
Monod Growth Kinetics
•The most widely used expression for describing specific growth rate as a function of substrate
concentration is attributed to Monod (1942, 1949)
•Relates specific growth rate, m, to substrate concentration
•Empirical---no theoretical basis—it just “fits”!
•Have to determine mmax and Ks in the lab
•Each m is determined for a different starting S

 max S

Ks  S

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Monod Growth Kinetics
• is the maximum specific growth rate observed and is the substrate
conversion corresponding to 1/2
Monod Growth Kinetics
• There are two constants in the equation µmax the maximum specific
growth rate and Ks the half saturation constant.
• Both are intrinsic physiological properties of particular type of
microorganisms.
• They also depend on substrate being utilized and temperature
of growth.
 Continuous Reactors

• Chemostat -- continuous stirred tank bioreactor for the cultivation of cells (CSTR )
• mixing supplied by impellers and rising gas bubbles
• assume complete mixing - composition of any phases do not vary with position
• liquid effluent has the same composition as the reactor contents

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Continuous Reactors
• The vessel that is used as a growth container in continuous culture is
called a bioreactor or chemostat
• Chemostat can produce microbial product more efficiently because
the chemostat can hold the exponential phase
Control Factors
• The growth and survival of bacteria depend on the close monitoring
and control of many conditions within the chemostat such as the pH
level, temperature, dissolved oxygen level, dilution rate, and agitation
speed.
• As expected with CSTRs, the pumps delivering the fresh medium and
removing the effluent are controlled such that the fluid volume in the
vessel remains constant
Control Factors - PH
• Different cells favor different pH environments.
• The designer needs to determine an optimal pH and maintain the
CSTR at it for efficient operation.
• Controlling the pH at a desired value during the process is extremely
important because under extreme pH conditions, cells cannot grow
properly, therefore appropriate action needs to be taken to restore
the original pH (i.e. adding acid or base).
Control Factors - Temperature
• Controlling the temperature is also crucial because cell growth
can be significantly affected by environmental conditions.
• Choosing the appropriate temperature can maximize the cell
growth rate as many of the enzymatic activates function the
best at its optimal temperature due to the protein nature of
enzymes.
Control Factors – Dilution Rate
• One of the important features of the chemostat is that it allows the operator to
control the cell growth rate.
• The most common way is controlling the dilution rate, although other methods
such as controlling temperature, pH or oxygen transfer rate can be used.
• Dilution rate is simply defined as the volumetric flow rate of nutrient supplied
to the reactor divided by the volume of the culture (unit: time -1).
• While using a chemostat, it is useful to keep in mind that the specific growth
rate of bacteria equals the dilution rate at steady state. At this steady state, the
temperature, pH, flow rate, and feed substrate concentration will all remain
stable.
• Similarly, the number of cells in the reactor, as well as the concentration of
reactant and product in the effluent stream will remain constant.
Control Factors - Dilution Rate

• It is essential to obtain the condition of steady state operation and to

determine the critical dilution rate, the maximum operable dilution
rate, to obtain an expression for cell productivity in a continuous
reactor/chemostat.
• Negative consequences can occur if the dilution rate exceeds the
specific growth rate.
Washout
• when the dilution rate is greater than the specific growth rate (D > μ), the dCC/dt term becomes
negative.
𝑑𝐶𝐶/𝑑𝑡=(𝜇−𝐷)𝐶𝐶
• This shows that the concentration of cells in the reactor will decrease and eventually become zero.
• if a dilution rate is chosen that is higher than μmax, the cells cannot grow at a rate as fast as the rate
with which they are being removed so the culture will not be able to sustain itself in the bioreactor,
and will wash out.
• This is called wash-out, where cells can no longer maintain themselves in the reactor.
• Therefore the dilution rate at which wash-out will occur:

𝐷𝑚𝑎𝑥=
Washout
• So, an operational parameter F for a given V determines a biological
parameter that is the significance of this equation.
• So, that will happen under steady state conditions of operation of a
chemostat.
• So, if you want the cells to grow at a higher rate you just have to
increase the flow rate and the cells will grow at a higher rate and till a
certain point.
• If you turn knobs incorrectly: if D is too high, the cells cannot max
grow fast enough to reach steady-state. Washout will occur.
Critical Dilution Rate
• Initially, the rate of cell production increases as dilution rate increases.
• When Dmaxprod is reached, the rate of cell production is at a maximum.
• This is the point where cells will not grow any faster. D = μ (dilution rate = specific
growth rate) is also established at this point, where the steady-state equilibrium is
reached.
• The concentration of cells (CC) starts to decrease once the dilution rate exceeds the
Dmaxprod.
• The cell concentration will continue to decrease until it reaches a point where all
cells are washed out.
• At this stage, there will be a steep increase in substrate concentration because
fewer and fewer cells are present to consume the substrate.
Critical Dilution Rate
• Since the concentration of the limiting nutrient in the chemostat
cannot exceed the concentration in the feed.
• The specific growth rate that the cells can reach in the chemostat
is usually slightly lower than the maximal specific growth rate
because specific growth rate usually increases with nutrient
concentration as described by the kinetics of the Monod Equation.
•  The highest specific growth' rates (μmax) cells can attain is equal to
the critical dilution rate (Dcrit)
• The dilution rate at which x=o is the critical dilution rate Dcrit and
must not be exceeded
Critical Dilution Rate