4th year class ppt bio.2014 final .pdf

Bahirdar university
Bahirdar institute of technology /BiT
Faculty of chemical and food engineering
Fundamental of Biochemical Engineering /4122
2013 EC
By Ins. Birhanu Ayitegeb

CONTENTS OF THE COURSE
1. Introduction
2. Enzyme kinetics
3. Immobilized Enzyme
4. Cell Kinetics and Fermenter Design
5. Sterilization
6. Agitation and Aeration
7. Bioprocess control
8. Biosensors
9. Downstream Processing

Course Description : summary
This course focuses on the interaction of chemical engineering,
biochemistry, and microbiology.
Mathematical representations of microbial systems are featured
among lecture topics.
 Kinetics of growth, death, and metabolism are covered. Continuous
fermentation, agitation, mass transfer, and scale-up in fermentation
systems, and enzyme technology complete out the subject material.

Course Objectives : summary
To provide the students with the fundamental understanding of
fundamental biochemical engineering so that the students are
capable of functioning effectively as an industrial Biochemical
engineer or prepare for academic research

General Topics to Be Covered
Fermentation Engineering
• Microbial Cultures （Media，Sterilization）
• Microbial Growth （Growth Kinetics）
Enzyme Technology
• Enzyme kinetics with Structured and Unstructured models
• Immobilized enzymes
• Application of Enzymes
Bioreaction Engineering
• Batch and continuous study
• Fed-batch study
Reactor design and modeling
• Fermenter design
• Agitation and mixing
Downstream Processing in Biochemical Engineering
• Purification
• Product recovery

Introduction
• What is Biochemical Engineering?
• Development of Biochemical Engineering
• Biology and Engineering Study in Biochemical Engineering
• Application of Biochemical Engineering

Biochemical Engineering
 The most basic definition of biochemical engineering is the application of
chemical engineering principles to biological systems.
The application of chemical engineering principles :
To design, develop, operate, or utilize processes and products based on
biological and biochemical phenomena
 this field is included in a wide range of industries, such as health care,
agriculture, food, enzymes, chemicals, waste treatment, and energy.

 Biochemical engineering is a branch of chemical engineering that
mainly deals with the design and construction of unit processes that
involve biological organisms or molecules.
Biochemical engineering is concerned with conducting biological
processes on an industrial scale.
This area links biological sciences with chemical engineering.

 Application of biological sciences in industrial processes is known as bioprocessing.
The role of biochemical engineers has become more important in recent years due to the
dramatic developments of biotechnology.
What is Biotechnology?
 Biotechnology can be broadly defined as “
Commercial techniques that use
living organisms, or substances from those organisms, to make or modify a
product, including techniques used for the improvement of the characteristics
of economically important plants and animals and for the development of
microorganisms to act on the environment …
”(Congress of the United
States, 1984).


Biochemical engineering is interdisciplinary course. The relationship between Biochemical
Engineering and its three related fundamental subjects.

The task of Biochemical Engineering
Development of an economical process to maximize biomass production
(and hence a particular chemical, biochemical, or protein), taking into
consideration raw-material and other operating costs.
Typical biological process

The task of Biochemical Engineering
1. to obtain the best biocatalyst (microorganism, animal cell, plant cell, or
enzyme) for a desired process.
2. to create the best possible environment for the catalyst to perform by
designing the bioreactor and operating it in the most efficient way.
3. to separate the desired products from the reaction mixture in the most
economical way
4. Cultivation of plant cells, insect cells, and mammalian cells, as well as the
genetically engineered versions of these cell types.
5. Use of the tools of molecular genetics, often coupled with quantitative
models of metabolic pathways and bioreactor operation, to optimize cellular
function for the production of specific metabolites and proteins.
6. Identification, design, and use of biocatalysts for the production of useful
chemicals and biochemicals

Development of Biochemical Engineering
Early recognition of fermentation phenomena
• In 1680, invention of microscope ─ existence of microorganisms
• In 1857, Pasteur found that alcohols was produced by yeast
• In 1897, Edward Buchner demonstrated that the fermentation of carbohydrates
results from the action of different enzymes contained in yeast and not the yeast
cell itself.

• At the end of 19th century, anaerobic fermentation, such as production of
alcohol, lactic acid and food using yeast and Lactobacillus
• At early 20th century，acetone-butanol was produced by Clostridium aceto-
butylicum in 1916 in England. Glycerol was produced using sulfite as substrate
in Germany ─ development of food industry to non-food industry.
• Aerobic fermentation technology: air was supplied to produced bioproducts
such as acetic acid, yeast cells.
• In 1933, shake flask culture technology

• At early 1940s，the production of penicillin
• In 1928, Fleming found penicillin
• In 1941, USA cooperated with England to study the production of penicillin
• Surface culture：1 L flask was filled with 200 mL buckwheat medium ─ 40
U/mL
• In 1943, submerged culture： 5 m3 ─ 200 U/mL
• Today：200 m3 ─ 100 m3 ─ 5-7104 U/ml
• Other antibiotics: streptomycin, aureomycin, neomycin, and Chlortetracycline,
etc.

Stirrer of large-scale fermentor

180 m3 fermentor plant
Large air compressor

Modern Biotechnology ─ Molecular Biology and Fermentation Engineering
Amino acid fermentation technology ─ glutamic acid, lysine
Nucleic acid fermentation industry ─ Inosinic acid, and
Guanosine 5’-triphosphate (GTP)
 Metabolic control of mutant ─ metabolic control fermentation
technology
 Break-down by-pathway: Control of enzyme activity, control of
enzyme synthesis

Application of Biochemical Engineering
•Food industry
• Enzyme Industry
• Amino acid Industry
• Organic acid Industry
• Feedstuff Industry
• Development of new materials
• Environmental Protection

Application in food industry
Earliest applications of Biochemical Engineering:
Alcohol contained drink：wine, wine, distilled spirit, beer, aquavit, whiskey,
barley-bree, vodka, champagne, etc.
Traditional condiment: catsup, soy sauce, vinegar, lobster sauce, preserved
beancurd, tofu, pickled vegetable, etc.
Fermented dairy product：milk, cheese, yoghourt, kefir, etc.

Application in food industry
Food materials produced by using fermentation or enzyme technology:
glucose, maltose, fructose – glucose syrup, mannitol, etc.
Food additives: baker yeast, monosodium glutamate, lysine, citric acid, red
rice (pigment), sweet peptide, inosinic acid, (IMP) and guanylic acid (both are
flavour-enhancers ), glucose oxidase, etc.
Novel fermented drink: active lactic acid drink.

Application in medicine industry
A . Different antibiotics
B . Amino acids：amino acid transfusion
C .Vitamins
D. Steroid hormones
E. Bioproducts: Prevention, diagnosis and treatment of infectious
diseases
F. Monoclonal antibodies: preparation of diagnosis kits, disease therapy,
strain identification, etc.
G. Others：Enzymes for therapy, enzyme inhibitors, nucleic acids,
enzymes for medicine industry, etc.

Antibiotics
 About 400 antibiotics are produced by fermentation, among which
about 120 were commonly used. In recent years, the global sale of
antibiotics exceeded US$40 billion.

Application in enzyme industry
• Sugar enzymes:  -amylase, β-amylase, saccharifying enzyme,
amylopectase, invertase, isomerase, cellulase, and galactase
• Protease: alkali, acidic and neutral protease
• Pectinase: an enzyme that catalyzes the hydrolysis of pectin to pectic
acid and methanol.
• Lipase: any of a group of enzymes that catalyze the hydrolysis of fats
into glycerol and fatty acids.
• Chymosin: an enzyme that induces coagulation
• Catalase: an enzyme that catalyzes the decomposition of hydrogen
peroxide into water and oxygen.

Application in enzyme industry
-amylase (top left) , pectinase (top right),
catalase (bottom)

Application in energy industry
Using fermentation, biotransformation and enzyme to produce marsh gas,
hydrogen energy, alkane, ethanol, solvents, organic acids, biodiesel, and
polysaccharides, etc.
Biofuel, such as ethanol, plays an important role in new energy source.
Biofuel

Application in feedstuff industry
• Production of proteins for feedstuff.
The deficiency of proteins is rather serious with the increasing population.
• Microorganism cells contain 45-55% protein in respect to dry cell mass, thus
production of the single cell protein (SCP) by fermentation is the important
protein source for food an

Application in agriculture
• Antibiotics
• Single cell protein (SCP)
• Fungi for food and medicine
• Biopesticides
• Biofertilizer

Environmental Protection
Elimination of petroleum pollution by
genetically engineered bacteria

Microorganisms and environmental protection

Biological and Engineering study of
Kinetics and Bioreactor engineering: Bioreaction Engineering
Intrinsic kinetics：Kinetics that represent the maximum capability of the
members of the microbial community with the fastest growth kinetics,
without influence of different transport processes in bioreactor.
Bioreactor Engineering deals with the form, structure, operation mode and
transport phenomena of bioreactors, which may influence the macrokinetics
of microorganisms

Biological and Engineering study of
Computer control of fermentation process
Computer control systems：System-on-chip (SOC), Industrial Personal
Computer (IPC), programmable Logic Controller (PLC), Distributed Control
System (DCS)…
Sensors：DO, pH, waste air analysis system …
 Parameters automatic control circuit
 Automatic control ： Automatic control of temperature ， air supply,
bioreactor pressure, pH, DO and rotation speed, and so on

Fermentors
Central Control Unit of a fermentation plant

Central Control Unit of a fermentation plant

Assignments for assessment
• A literature based assignment will be given to the students within the course
which needs to be submitted at the end of the lecture series. The assignment
needs to be done per each group of students. [weight = 20%]
Literature-based Assignment
 Recovery of Baker’s yeast (group 1)
 Production of monosodium glutamate (group 2)
 Recovery of an extracellular enzyme (group 3)
 Recovery of intracellular enzyme (group 4)
 Recovery of single-cell protein on a methanol basis (group 5)
 Recovery of toxoid vaccines (group 6)
 Recovery of whole-cell vaccines (group 7)

Assignment detail
 Using a small industrial example (from literature), prepare a clear flow
sheet of the process (to be treated as a state-of-the-art)
 State the function of the different important unit operations that are
involved
 Mention the variables that can influence the system
 Can you simplify the flow sheet ? If yes, why and how ? Be critical in your
reply.

Enzyme
INTRODUCTION :
 Enzymes are biological catalysts that are protein molecules in
nature.
 They are produced by living cells (animal, plant, and
microorganism) and are absolutely essential as catalysts in
biochemical reactions.
 Almost every reaction in a cell requires the presence of a specific
enzyme.
 A major function of enzymes in a living system is to catalyze the
making and breaking of chemical bonds.
 Therefore, like any other catalysts, they increase the rate of reaction
without themselves undergoing permanent chemical changes.

INTRODUCTION :
 The catalytic ability of enzymes is due to its particular protein structure.
A specific chemical reaction is catalyzed at a small portion of the surface of an
enzyme, which is known as the active site.
 Enzyme reactions are different from chemical reactions, as follows:
1. An enzyme catalyst is highly specific, and catalyzes only one or a small
number of chemical reactions.
2. The rate of an enzyme-catalyzed reaction is usually much faster than that of
the same reaction when directed by nonbiological catalysts.
3. The reaction conditions (temperature, pressure, pH, and so on) for the enzyme
reactions are very mild.
4. Enzymes are comparatively sensitive or unstable molecules and require care
in their use.

INTRODUCTION
 Enzymes bind temporarily to one or more of the reactants of the reaction they
catalyze. In doing so, they lower the amount of activation energy needed and thus
speed up the reaction.

INTRODUCTION :
 Enzymes as Biological Catalysts
Increase reaction rates by over 1,000,000-fold
 Two fundamental properties
 Increase the reaction rate with no alteration of the
enzyme
 Increase the reaction rate without altering the
equilibrium
 Reduce the activation energy

INTRODUCTION :
Enzymes as Biological Catalysts
The substrate binds to a specific region called the active site

Enzyme Kinetics
• Study of the rates of enzyme-catalyzed reactions
• Provides information on enzyme specificities and mechanisms

Enzyme Kinetics
EnzymeInhibition
 a substance which can combine with enzymes to alter their catalytic
activities.
 Many different kinds of molecules inhibit enzymes and act in a
variety of ways.
 One major distinction is whether the inhibition is
1. Reversible
 Competitive with substrate or
 Uncompetitive inhibitors
 Non-Competitive with the substrate
2. Irreversible (It is covalently bound, incapacitating the enzyme)

Example
Evaluate the Michaelis-Menten kinetic parameters by employing the
Lineweaver-Burk plot, the Eadie-Hofstee plot, and regression technique

Chapter three
Immobilized Enzyme

Immobilized Enzyme
Introduction:
 Since most enzymes are globular protein, they are soluble in water. Therefore, it is very
difficult or impractical to separate the enzyme for reuse in a batch process.
Immobilization is defined as the localization of cell or enzyme in a distinct support or
matrix
The support or matrix on which the enzyme are immobilized allow the exchange of
medium contusing substrate or effector or inhibitor molecules.
Immobilization enhances the stability of enzyme.

Advantages of Enzyme Immobilization

Immobilized Enzyme
Properties of material which used as matrix or supports
Inert/Non reactive
Physically strong and stable
Should be cheap, enough
Regenerated
Cost effective
Easily available
Surface available

 Immobilization techniques can be classified by basically two methods,
 chemical and
 physical method.
Chemical methods:
Covalent Attachment:
Cross-linking Using Multifunctional Reagents
Physical methods
 Adsorption:
Ionic Binding
 Entrapment:
 Microencapsulation:
 Covalent attachment : Commonly employed water-insoluble supports for the covalent attachment
of enzymes include:
synthetic supports such as acrylamide-based polymers, maleic anhydride-based
polymers, methacrylic acid-based polymers, styrene-based polymers, and
polypeptides, and
natural supports such as agarose (Sepharose), cellulose, dextran (Sephadex), glass,
and starch
.

Cross linking using multi functional reagents

 Adsorption :
 This method is the simplest way to immobilize enzymes. Enzymes can be adsorbed
physically on a surface-active adsorbent by contacting an aqueous solution of enzyme with
an adsorbent.
Van der Waals
Carriers: silica, carbon nanotube, cellulose, etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected

Entrapment:
 Enzymes can be entrapped within cross-linked polymers by forming a highly
cross-linked network of polymer in the presence of an enzyme.
 This method has a major advantage in the fact that there is no chemical
modification of the enzyme, therefore, the intrinsic properties of an enzyme
are not altered.
However, the enzyme may be deactivated during the gel formation.
 Enzyme leakage is also a problem.
The most commonly employed cross-linked polymer is the polyacrylamide
gel system.
This has been used to immobilize alcohol dehydrogenase, glucose oxidase,
amino acid oxidase, hexokinase, glucose isomerase, urease, and many other
enzymes.

 Microencapsulation:
 Enzymes can be immobilized within semipermeable membrane microcapsules.
This can be done by the interfacial polymerization technique.
 Organic solvent containing one component of copolymer with surfactant is
agitated in a vessel and aqueous enzyme solution is introduced.
The polymer membrane is formed at the liquid-liquid interface while the
aqueous phase is dispersed as small droplets.
 One example of this process is the polyamide nylon system, in which 1,6-
diaminohexane is the water-soluble diamine and 1,10-decanoyl chloride is the
organic-soluble diacid halide.
 The organic solvent for this system is a chloroform-cyclohexane mixture.
 The immobilized enzyme produced by this technique provides an extremely
large surface area.

Effect of Mass-Transfer Resistance
The immobilization of enzymes may introduce a new problem which is absent free soluble
enzymes.
It is the mass-transfer resistance due to the large particle size of immobilized enzyme or due
to the inclusion of enzymes inpolymeric matrix.
 If we follow the hypothetical path of a substrate from the liquid to the reaction site in an
immobilized enzyme, it can be divided into several steps (1) transfer from the bulk liquid to
a relatively unmixed liquid layer surrounding the immobilized enzyme; (2) diffusion
through the relatively unmixed liquid layer; and (3) diffusion from the surface of the
particle to the active site of the enzyme in an inert support.
Steps 1 and2 are the external mass-transfer resistance. Step 3 is the internal particle mass
transfer resistance.

 If an enzyme is immobilized on the surface of an insoluble particle,
the path is only composed of the first and second steps, external mass-
transfer resistance.
The rate of mass transfer is proportional to the driving force, the
concentration difference, as
where CSb and Cs are substrate concentration in
the bulk of the solution and at the immobilized
enzyme surface, respectively
The term Ks is the mass-transfer coefficient
(length/time)
and A is the surface area of one immobilized
enzyme particle.

 During the enzymatic reaction of an immobilized enzyme, the rate
of substrate transfer is equal to that of substrate consumption.
Therefore, if the enzyme reaction can be described by the
MichaelisMenten equation,
where a is the total surface area per unit
volume of reaction solution.

Cont …
The limiting factor is
enzymatic rxn
The limiting factor here is
subsrate concentration

Limitations of Immobilized of Enzymes

Chapter four
Cell Kinetics and Fermenter Design

Growth Cycle for Batch Cultivation

Various Models for Cell Kinetics
types of
culture vessel:

types of culture vessel
 There are two types of culture vessel: chemostat and biostat;
(i) Chemostat (growth rate controlled by dilution rate, D, (h1)
(ii) Turbidostat (constant cell density that is controlled by the fresh medium)
Chemostat.
The nutrients are supplied at a constant flow rate and the cell density is adjusted with the
supplied essential nutrients for growth.
In a chemostat, growth rate is determined by the utilisation of substrates like carbon, nitrogen
and phosphorus.
Turbidostat .
 It is a system where cell growth is controlled and remains constant while the flow rate of
fresh media does not remain constant.
Cell density is controlled based on set value for turbidity, which is created by the cell
population while fresh media is continuously supplied.
 Chemostat (growth rate controlled by dilution rate, D, (h1)
 Turbidostat (constant cell density that is controlled by the fresh medium)

Exponential Growth Phase
 In unicellular organisms, the progressive doubling of cell number results in a
continually increasing rate of growth in the population.
 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:
………………………………………………………………………………………………………1
…………………………………………………………………………………………2

Bioreactor Design
 The bioreactor is the heart of any biochemical process in which enzymes, microbial, mam-
malian or plant cell systems are used for manufacture of a wide range of useful biological
products.
The performance of any bioreactor depends on many functions, such as those listed below:
Biomass concentration: must remain high enough to show high yield
Nutrient supply
 Sterile conditions : must be maintained for pure culture system.
Product removal
Effective agitations: is required for uniform distribution of substrate and microbes in the
working volume of the bioreactor.
Product inhibition
 Heat removal: is needed to operate the bioreactor at constant temperature, as the desired
optimal microbial growth temperature
Aeration
Correct shear conditions : High shear rate may be harmful to the organism and disrupt the cell wall; low shear
may also be undesirable because of unwanted floccula-tion and aggregation of the cells, or even growth of bacteria on
the reactor wall and stirrer.:
Metabolisms/microbial activities

Cont…

From eqution 1
……………………………… 3
…………………………… 4

operation modes:
- batch: stirred tank
- continuous: chemostat, fluidized-bed
- modified types of the above modes:
fed-batch, chemostat with recycle,
multi-stage continuous reactors

Disadvantages of Batch Culture
 The nutrient in the working volume becomes depleted;
 limitation and depletion of the substrate.
Since there is no flow stream to take effluent out, as the system is closed,
toxins form there.
Death phase quickly in an old culture.
The long duration of the batch system for slow growth results in exhaustion
of essential nutrients and an accumulation of metabolites as byproducts.
Exhaustion of nutrients and substrate may cause the system to become
retarded.
The technical problem resulting in changes to media composition may
directly affect the microbial exponential growth phase.
Product inhibition may block enzyme activities, and the cells became
poisoned by the by-product.
the product should be sent for downstream processing, then
the system has to be cleaned and recharged with fresh feed, so the process is
highly labour intensive for downtime and cleaning.

Advantages of Continuous Culture
 all the problems associated with the batch culture are solved.
 Firstly, the growth rate is controlled and the cells are well maintained, since fresh
media is replaced by old culture while the dilution is taking place.
 the effect of physical and chemical parameters on growth and product formation can
easily be examined.
The biomass concentration in the cultured broth is well maintained at a constant
dilution rate.
The continuous process results in substrate-limited growth and cell-growth-limiting
nutrients.
The composition of the medium can be optimised for maximum productivity; in
addition secondary metabolite production can also be contro
The growth kinetics and kinetic constants are accurately determined.
The process leads to reproducible results and reliable data. High productivity per unit
volume is achieved.
The continuous culture is less labour-intensive, and less downtime is needed.
Finally, steadystate growth can be achieved, even if mixed cultures are implemented.

TYPE OF BIOREACTOR
1. Airlift Bioreactors
 mixing is accomplished without any mechanical agitation.
 used for tissue culture because the tissues are shear sensitive and normal mix-
ing is not possible.
air is fed into the bottom of a central draught tube through a sparger ring, so
reducing the apparent density of the liquid in the tube relative to the annular
space within the bioreactor.
 The flow passes up through the draught tube to the head space of the
bioreactor, where the excess air and the by-product, CO2, disengage.
Cooling can be provided by either making the draught tube an internal heat
exchanger or with a heat exchanger in an external recirculation loop.
The advantages of airlift bioreactor are:1. In low shear, 2. sterility is easily
maintained.3. the pressure at the bottom ofthe vessel will increase the oxygen
solubility, 4. Extremely large vessels can be constructed

TYPE OF BIOREACTOR
2. Airlift Pressure Cycle Bioreactors
The gas is circulated by means of pressurized air.
In airlift bioreactors, circulation is caused by the motion of injected gas
through a central tube,
3. Loop Bioreactor
A modified type of airlift system with gas and liquid flow patterns in which a
pump trans-ports the air and liquid through the vessel
 Gas and circulated liquid are injected into the tower through a noz-zle.
4. STIRRED TANK BIOREACTORS
The most important bioreactor for industrial application is the conventional
mixing vessel,
which has the dual advantages of low capital and low operating costs.

DESIGN EQUATIONS FOR CSTR FERMENTER
 In designing a bioreactor, material balance is used for all the streams
associated with the fermentation vessel.
The biomass at inlet, outlet and the generated biomass must be bal-anced while
the fermentation proceeds. The cell balance without any cell accumulation is
shown in the following equation:
.

DESIGN EQUATIONS FOR CSTR FERMENTER
• The cell balance without any cell accumulation is shown in the following
equation:
where X is viable cell in the effluent stream
 X0 is viable cell in the feed stream,
F is the volumetric flow rate,
V is the reactor working volume,
rx is the rate of cell formation pe runit volume.
The rate equation is explained in detail by a Monod rate model.
The Monod rate equation is well known in microbial growth kinetics:

Monod Model for a Chemostat
A Monod rate model is used to demonstrate the rate of biomass generation.
We neglect the cell death rate.
Let us denote the ratio of biomass rate of generation to biomass concentra-tion,rx/X,
that is the specific growth rate; μ also denotes the dilution rate; D is defined as number
of tank volumes passed through per unit time,F/V. After substitution of D and μ into
above equation , the following equation is obtained

……………7
………………..6

 When the volume of the vessel is divided by the flow rate, retention time and dilution rate
are defined in the following equation:
………………………….. 8
……..…………………………… 9
…………………………………..10

……………………………………….11
…………………………………………. 12
………………………………..13

………………………………….. 14
…………………………15
……………………………………….16

• Near the wash out, the reactor is very sensitive to variations of dilution
rate D. A small change in D gives a relatively large shift in X and S..
………………………………………17

Example
Find also the retention time and the final out put of substrate concentration
of substrate ?

INTRODUCTION
Sterilisation is the action of eliminating microorganisms from a medium.
Sterility is the absence of any detectable and viable microbes in a culture
medium or in the gas phase.
Sterilisation is a process that destroys all living organisms, spores and viruses
in a pressurized vessel at high temperature.
 In the food and dairy industries, sterilisation is commonly used to preserve
food products.
At the laboratory scale, huge steel vessels with live stream at 105 kPa (15 psig)
are commonly used for 20–30 minutes.
This is a closed system known as an autoclave; therefore it is batch
sterilisation. The temperature is raised to 121 C
Even at high temperatures the fungal spores may survive if only heat is used.
Therefore, media is autoclaved at 121 C and 105 kPa (15 psig

INTRODUCTION
• Overheating the prepared media may have negative impact, causing the media
pH to be unstable.
• Acid pH is very sensitive to overheating.
• Overheating in media containing sugars causes the carbohydrate to be
caramelised.
• This media may have reverse impact, reducing bacteriological performance.
• Gelatinous media or any nutrient agar at acidic pH is hydrolysed.
• This is due to the acidic condition of catalytic activities or excess amounts of
protons
• breaking down the solid media and extra sugars forming. This may cause
substrate inhibition

BATCH STERILISATION
• Batch sterilisation uses steam to eliminate living organisms.
• Heat losses, heating and cooling are major steps, and it is a time-consuming
process.
• It requires air to be evacuated replaced with steam.
• At first the chamber is flashed with pressurised steam.
• supplying steam and maintaining the set pressure and temperature at a constant
level for a fixed duration.
• Batch sterilisation wastes energy and overcooks the medium.
• There is no conservation of energy; therefore the process may not be
economical for implementation at a large scale.
• Batch sterilisation is common at bench and laboratory scales

BATCH STERILISATION
 Batch media sterilisation is done in an autoclave.
Basically, it is a huge steam cooker.
• Steam enters the jacket of the surrounding chamber.
• When the pressure from the steam has been built up in the jacket, a venting
valve for the outlet of chamber air is closed and the inlet
• valve allows the steam to enter the chamber. The pressure of the chamber is
increased to 105 kPa (15 psig).
• At this point the sterilisation time begins to count down.
• Usually 15–30 minutes are used, depending on the type and volume of
media. times for sterilisation. There are recommended holding times of 20,
10 and 3 minutes
• for 121, 126 and 134 C, respectively.

CONTINUOUS STERILISATION
• One of the methods for continuous sterilization of medium for fermentation is
the direct use of live steam by injection of steam into the medium.
• The heat exchanger is eliminated.
• The medium stays in a loop for a predetermined holding time until the entire
medium is sterile.
• . To utilise all the supplied heat, preheaters or heat economisers are used.
• Instead of having a cold water stream to cool the sterile media, the lower
temperature,
• unsterile media stream takes the primary heat from the warm stream, cooling
the sterile media.
• Continuous sterilisation has a holding coil for detention long enough to kill all
the

CONTINUOUS STERILISATION
• The medium from a make-up vessel flowing through the exchanger is held in the coil,
and then goes through the heat exchanger, heating more unsterile medium while
become cool itself, as it is collected in a sterile fermenter. Heat economisers are
important for large-scale plant unit in continuous sterilisation.
• For a small-scale bioreactor use of direct steam injection is much simpler to operate. A
heat exchanger is then needed with cooling water to bring the medium back to
ambient temperature.
• Therefore jacketed vessels are commonly used with cooling water. There are
advantages and disadvantages to batch and continuous sterilisation. The energy
savings are related to the use of an economiser and direct stream or indirect
sterilisation.
• In a largescale system economy and role decide which one is the most suitable process
to be implemented.
• For each case the specific design needs to be evaluated in terms of the thermal
efficiency and fixed costs involved.

HOT PLATES
• Hot-plate heat exchangers are extremely efficient and easily maintained if any
fouling or scale deposition takes place.
• Normally fouling is serious problem, occur-ing by deposition of proteins on the
hot surface of the exchanger, which causes reduction in the overall heat transfer
coefficient.
• Plate exchangers are easily separated and cleaned.Special care must be taken
when the media contains any agglomerated particles.
• Correla-tion is required to calculate the suitable residence time for sterilisation.
The choice of a uitable sterilisation process is based on economics and cost of the
heat exchangers toreduce energy consumption.
• In a suitable exchanger, the larger the heat transfer coefficient,the greater the
energy recovery.

HIGH TEMPERATURE STERILISATION
• Fast sterilisation is performed at high temperature.
• High-temperature sterilisation requires short holding times.
• This technique is used in the fast preparation of nutrient media for industrial bioprocesses
and in pasteurising milk.
• A short retention time may favour media with heat-sensitive proteins and cause less damage
to the biochemical composition of the media than more prolonged times at lower
temperatures.
• The ability of high temperatures to perform rapid sterilisation is related to activation
energies.
• That is affected by how fast bacteria are killed in an elevated temperature.
• On the other hand, long sterilisation at high temperatures may destroy the protein and
biochemical composition of the media.
• Short-duration sterilisation at high-temperatures are more lethal to organisms and less
chemically damaging than are longer sterilisation processes at lower temperatures.
• Sterilisation at hightemperature is recommended for 3 minutes at 134 C.

STERILISED MEDIA FOR MICROBIOLOGY
• Sterile media are used for pure culture.
• The media used to culture microorganisms depend on the living conditions of
the microorganisms.
• The kinetics of culture media sterilisation describe the rate of destruction of
microor-ganisms by steam using a first-order chemical reaction rate model. As
the population of microorganisms (N) decreases with time, the rate is defined
by the following equation:
where N is the number of viable organisms present in the culture media,t is the
retention time or sterilisation time, and kd is the reaction rate constant as it is
known for a specific death rate. Using separation of variables and integrating
with initial condition, the follow-ing useful expression is obtained

Chapter six
Agitation and Aeration

Introduction
One of the most important factors to consider in designing a fermenter is the
provision for adequate mixing of its contents.
The main objectives of mixing in fermentation are
 to disperse the air bubbles, to suspend the microorganisms (or animal and plant
tissues), and
to enhance heat and mass transfer in the medium.
Since most nutrients are highly soluble in water, very little mixing is required
during fermentation just to mix the medium as microorganisms consume
nutrients.
Adequate oxygen supply to cells is often critical in aerobic fermentation.
 Even temporary depletion of oxygen can damage cells irreversibly.
Therefore, gaseous oxygen must be supplied continuously to meet the requirements for
high oxygen needs of microorganisms, and the oxygen transfer can be a major limiting
step for cell growth and metabolism.

Mass-Transfer Path
The path of gaseous substrate from a gas bubble to an organelle in a
microorganism can be divided into several steps
1. Transfer from bulk gas in a bubble to a relatively unmixed gas layer
2. Diffusion through the relatively unmixed gas layer
3. Diffusion through the relatively unmixed liquid layer surrounding the
bubble
4. Transfer from the relatively unmixed liquid layer to the bulk liquid
5. Transfer from the bulk liquid to the relatively unmixed liquid layer
surrounding a microorganism
6. Diffusion through the relatively unmixed liquid layer
7. Diffusion from the surface of a microorganism to an organelle in which
oxygen is consumed
Steps 3 and 5, the diffusion through the relatively unmixed liquid layers of
the bubble and the microorganism, are the slowest among those outlined as a
result, control the overall mass-transfer rate.

Mass-Transfer Path
 Steps 3 and 5, the diffusion through the relatively unmixed liquid layers of the
bubble and the microorganism, are the slowest among those outlined previously
and, as a result, control the overall mass-transfer rate.
 Agitation and aeration enhance the rate of mass transfer in these steps and
increase the interfacial area of both gas and liquid.
vital tools for the design and operation of fermenter systems are :
interfacial area,
bubble size,
gas hold-up,
agitation power consumption, and
volumetric mass-transfer coefficient

Molecular Diffusion in Liquids
 When the concentration of a component varies from one point to another, the
component has a tendency to flow in the direction that will reduce the local differences
in concentration.
Molar flux of a component A relative to the average molal velocity of all constituent JA is proportional
to the concentration gradient dCA /dz as:
which is Fick’s first law
•
 The DAB diffusivity of component A through B, which is a measure of its diffusive
mobility.
 Molar flux relative to stationary coordinate NA is equal to:
 where C is total concentration of components A and B and NB is the molar
flux of B relative to stationary coordinate.
 The first term of the right hand side is the flux due to bulk flow, and the second
term is due to the diffusion. For dilute solution of A,

Cont…
 Diffusivity:
The kinetic theory of liquids is much less advanced than that of gases.
Therefore, the correlation for diffusivities in liquids is not as reliable as that for
gases. Among several correlations reported, the Wilke-Chang correlation (Wilke
and Chang, 1955) is the most widely used for dilute solutions of
nonelectrolytes.
 When the solvent is water, Skelland (1974) recommends the use of the
correlation developed by Othmer and Thakar (1953).
Where :

Mass-Transfer Coefficient
 The mass flux, the rate of mass transfer qG per unit area, is proportional to a
concentration difference. If a solute transfers from the gas to the liquid phase, its
mass flux from the gas phase to the interface NG is
 where CG and CGi is the gas-side concentration at the bulk and the interface, respectively, . kG
is the individual mass-transfer coefficient for the gas phase and A is the interfacial area.
Similarly, the liquid-side phase mass flux NL is
 where kL is the individual mass-transfer coefficient for the liquid phase.

Cont…
 Since the amount of solute transferred from the gas phase to the interface must equal that
from the interface to the liquid phase

Mechanism of Mass Transfer
 Several different mechanisms have been proposed to provide a basis for a
theory of interphase mass transfer. The three best known are:
 the two-film theory,
the penetration theory, and
 the surface renewal theory.
 The two-film theory supposes that the entire resistance to transfer is contained in two
fictitious films on either side of the interface, in which transfer occurs by molecular
diffusion. This model leads to the conclusion that the mass-transfer coefficient kL is
proportional to the diffusivity DAB and Inversely proportional to the film thickness zf as:
 Penetration theory (Higbie, 1935)assumes that turbulent eddies travel from the bulk
of the phase to the interface where they remain for a constant exposure time te. The
solute is assumed to penetrate into a given eddy during its stay at the interface by a
process of unsteady-state molecular diffusion. This model predicts that the mass-
transfer coefficient is directly proportional to the square root of molecular diffusivity

Cont…
 Surface renewal theory (Danckwerts, 1951) proposes that there is an infinite range of ages for
elements of the surface and the surface age distribution function φ(t) can be expressed as
 where s is the fractional rate of surface renewal. This theory predicts that
again the mass-transfer coefficient is proportional to the square root of the
molecular diffusivity

Tools for the Design and Operation of Fermenter Systems are :

Volumetric mass transfer coefficient
 Mass-transfer coefficient is a function of physical properties and vessel
geometry. Because of the complexity of hydrodynamic in multiphase mixing.
It is common to obtain an empirical correlation for the mass-transfer
coefficient by fitting experimental data. The correlations are usually expressed
by dimensionless groups since they are dimensionally consistent and also
useful for scale-up processes.
Earlier studies in mass transfer between the gas-liquid phase reported the
volumetric mass-transfer coefficient kLa.
Since kLa is the combination of two experimental parameters, mass-transfer
coefficient and interfacial area, it is difficult to identify which parameter is
responsible for the change of kLa when we change the operating condition of a
fermenter.

Volumetric mass transfer coefficient
 Agitation of fermentation broth creates a uniform distribution of air in the media.
 Once you mix a solution, you exert an energy into the system.
 Increasing power input reduces the bubble size and this in turn increases the interfacial
area.
 Therefore the mass transfer coef-ficient would be a function of power input per unit
volume of fermentation broth, which is also affected by the gas superficial velocity.
 The general correlation is expected to be as follows:
 where KLa is the volumetric mass transfer coefficient in s1;
 a is proportionality factor,
 as a constant;
 Pg is the agitator power under gassing conditions in W;
 VL is the liquid volume with out gassing in m 3;
 vg is the gas superficial velocity in m/s;
 and y and z are empirical constants.

GAS HOLD-UP
 The most important property of air bubbles in the fermenters is their size.
 If the gas is dispersed into many small bubbles rather than a few large ones, more
interfacial area per unit volume results.
 Small bubbles have a slow rising velocity. Consequently, they stay longer
in contact with the liquid, which allows more time for oxygen to dissolve.
 The fraction of the fluid volume occupied by gas is called gas hold-up: that is, the volume
fraction of gas phase to total gas–liquid volume.
 Small bubbles lead to higher gas hold-up, which is defined by the following equation
 Gas hold-up is one of the most important parameters characterizing the hydrodynamics in a
fermenter. Gas hold-up depends mainly on the superficial gas velocity and the power
consumption, and often is very sensitive to the physical properties of the liquid
𝐻 =
𝑉𝑔
𝑉𝑙+𝑉𝑔
where H= Gas hold up,Vg volume of gas bubble ,Vl volume of liquid in m 3.

GAS HOLD-UP
 Gas Sparging with No Mechanical Agitation: In a two-phase system where the
continuous phase remains in place, the hold-up is related to superficial gas velocity Vs and
bubble rise velocity Vt (Sridhar and Potter, 1980)
Akita and Yoshida (1973) correlated the gas hold-up for the absorption of oxygen in various aqueous solutions
in bubble columns, as follows
Gas Sparging with Mechanical Agitation: Calderbank (1958) correlated gas hold-up for the gas-liquid
dispersion agitated by a flat-blade disk turbine impeller as
where 2.16×10−4 has a unit (m) and Vt = 0.265 m/s when the bubble size is in the range of 2 - 5 mm diameter.

Measurement of Interfacial Area
 gas-liquid interfacial area, which can be measured employing several techniques such as
photography, light transmission, and laser optics.
 The interfacial area per unit volume can be calculated from the Sauter-mean diameter D32
and the volume fraction of gas-phase H, as follows:
 The Sauter-mean diameter, a surface-volume mean, can be calculated by measuring drop
sizes directly from photographs of a dispersion according to the following equation
 Photographic measurement of drop sizes is the most straightforward method among many
techniques because it does not require calibration. However, taking a clear picture may be
difficult, and reading the picture is tedious and time consuming. Pictures can be taken
through the base or the sidewall of a mixing vessel.

Measurement of Interfacial Area
Alderbank (1958) also correlated the Sauter-mean diameter for the gas-liquid
dispersion agitated by a flat-blade disk turbine impeller as follows

Characterization of agitation
 Using Reynolds number /Re
 As mixing is a complex process, the variables involved are considered together in
dimensionless group known as the Reynolds number ( Re). Re is used to characterize the
behavior of flow
where Di is the impeller diameter in m, N is the rotational speed of impellers in round per sec-
ond (rps), r is the fluid density in kg/m 3, and m is the viscosity of the fluid in kg m -1 s-1.
Fully turbulent flow exists above a Reynolds number of 10 4, whereas fully laminar flow
exists below 100; in between is the transitional region.
 Using Froude number
 Another group of dimensionless variables that are used to characterise mixing in a vessel is
the Froude number (Fr), which takes gravitational forces into account:

Characterization of agitation
 Using power number
 A third group, which is related to energy required by the agitator, is the power number.
 This shows the power consumption for stirring.
 The power consumption is related to fluid properties, the density and viscosity of the fluid,
the stirrer rotation rate and the impeller diameter.
 The power in the turbulent region is proportional to Ni3Di5; therefore the power number is
the ratio of power for the aerated fluid and the non-aerated powered system, which is

Power Consumption
 Power consumption by agitation is a function of physical properties, operating condition,
and vessel and impeller geometry. Dimensional analysis provides the following relationship
 The dimensionless group in the left-hand side of Eq. is known as power number NP,
 Froude number NFr, which takes into account gravity forces. The gravity force affects the
power consumption due to the formation of the vortex in an agitating vessel. The vortex
formation can be prevented by installing baffles.
 For fully baffled geometrically similar systems, the effect of the Froude number on the
power consumption is negligible

Power consumption
 The power number decreases with an increase of the Reynolds number and reaches a
constant value when the Reynolds number is larger than 10,000. At this point, the power
number is independent of the Reynolds number.
 For the normal operating condition of gas-liquid contact, the Reynolds number is usually
larger than 10,000. For example, for a 3-inch impeller with an agitation speed of 150 rpm,
the impeller Reynolds number is 16,225 when the liquid is water.
 The power required by an impeller in a gas sparged system Pm is usually less than the
power required by the impeller operating at the same speed in a gas-free liquids Pmo.
 The Pm for the flat-blade disk turbine can be calculated from Pmo (Nagata, 1975), as
follows:

Correlation of power number with Reynolds number

Scale-Up
Similitude :
 For the optimum design of a production-scale fermentation system (prototype), we must
translate the data on a small scale (model) to the large scale. The fundamental requirement for
scale-up is that the model and prototype should be similar to each other.
 Two kinds of conditions must be satisfied to insure similarity between model and prototype.
They are:
1. Geometric similarity of the physical boundaries: The model and the prototype must be the
same shape, and all linear dimensions of the model must be related to the corresponding
dimensions of the prototype by a constant scale factor.
2. Dynamic similarity of the flow fields: The ratio of flow velocities of corresponding fluid
particles is the same in model and prototype as well as the ratio of all forces acting on
corresponding fluid particles. When dynamic similarity of two flow fields with geometrically
similar boundaries is achieved, the flow fields exhibit geometrically similar flow patterns.

Cont…
 For example, if forces that may act on a fluid element in a fermenter during
agitation are the viscosity force FV, drag force on impeller FD, and gravity
force FG, each can be expressed with characteristic quantities associated with
the agitating system.
 According to Newton’s equation of viscosity, viscosity force is
The drag force FD can be characterized in an agitating system as
Since gravity force FG is equal to mass m times gravity constant g,
The summation of all forces is equal to the inertial force FI as,

Cont…
 Then dynamic similrity between a model (m) and a prototype (p) is achieved if:
or in dimensionless forms:

Cont…
Therefore, using dimensionless parameters for the correlation of data has
advantages not only for the consistency of units, but also for the scale-up
purposes.

Example
1. The mass transfer coefficient kL of oxygen transfer in fermenters is a function of Sauter mean diameter D32,
diffusivity DAB, and density ρc viscosity μc of continuous phase (liquid phase). Sauter-mean diameter D32 can
be calculated from measured drop-size distribution from the following relationship,
Determine appropriate dimensionless parameters that can relate the mass
transfer coefficient by applying the Buckingham-Pi theorem.

Example 2
2. The power consumption by an agitator in an un baffled vessel can be expressed as

Solution
If you use the same fluid for the model and the prototype, ρp = ρm and μp =
μm. Canceling out the same physical properties and s yield

Cont…
 which is conflicting with the previous requirement for the equality of the Reynolds
number. Therefore, it is impossible to satisfy the requirement of the dynamic similarity
unless you use different fluid systems. If to satisfy , the following
relationship must hold.
Therefore, if the kinematic viscosity of the prototype is similar to that of water, the
kinematic viscosity of the fluid, which needs to be employed for the model, should be
1/31.6 of the kinematic viscosity of water. It is impossible to find the fluid whose kinematic
viscosity is that small. As a conclusion, if all three dimensionless groups are important, it is
impossible to satisfy the dynamic similarity.

Cont …
 The previous example problem illustrates the difficulties involved in the scale-up of the findings of small-
scale results. Therefore, we need to reduce the number of dimensionless parameters involved to as few as
possible, and we also need to determine which is the most important parameter, so that we may set this
parameter constant. However, even though only one dimensionless parameter may be involved, we may
need to define the scale-up criteria.
As an example, for a fully baffled vessel when ReN >10,000, the power
number is constant . For a geometrically similar vessel, the dynamic similarity
will be satisfied by

Rule of scale up
 There is no one scale-up rule that applies to many different kinds of mixing operations.
Theoretically we can scale up based on geometrical and dynamic similarities, but it has been shown that it is
possible for only a few limited cases. However, some principles for the scale-up are as follows (Oldshue,
1985):
1. It is important to identify which properties are important for the optimum operation of a mixing system. This
can be mass transfer, pumping capacity, shear rate, or others. Once the important properties are identified, the
system can be scaled up so that those properties can be maintained, which may result in the variation of the less
important variables including the geometrical similarity.
2. The major differences between a big tank and a small tank are that the big tank has a longer blend time, a
higher maximum impeller shear rate, and a lower average impeller shear rate.
3. For homogeneous chemical reactions, the power per volume can be used as a scale-up criterion. As a rule of
thumb, the intensity of agitation can be classified based on the power input per 1,000 gallon as shown in Table 9.4

Rule of scale up
4. For the scale-up of the gas-liquid contactor, the volumetric mass-transfer coefficient kLa can be used as a
scale-up criterion. In general, the volumetric mass-transfer coefficient is approximately correlated to the power
per volume. Therefore, constant power per volume can mean a constant kLa.
5. Typical impeller-to-tank diameter ratio DI/T for fermenters is 0.33 to 0.44. By using a large impeller,
adequate mixing can be provided at an agitation speed which does not damage living organisms. Fermenters are
not usually operated for an optimum gas-liquid mass transfer because of the shear sensitivity of cells, which is
discussed in the next section.

Shear-Sensitive Mixing
 One of the most versatile fermenter systems used industrially is the
mechanically agitated fermenter.
 This type of system is effective in the mixing of fermenter contents, the
suspension of cells, the breakup of air bubbles for enhance oxygenation, and
the prevention of forming large cell aggregates.
 However, the shear generated by the agitator can disrupt the cell membrane
and eventually kill some microorganisms animal cells, and plant cells .
As a result, for optimum operation of an agitated fermentation system,
we need to understand the hydrodynamics involved in shear sensitive mixing.
 For the laminar flow region of Newtonian fluid, shear stress τ is equal to the
viscosity µ times the velocity gradient du/dy as

Cont…
 which is known as Newton’s equation of viscosity. The velocity gradient is also known as
shear rate .
 For the turbulent flow,
is eddy viscosity, which is not only dependent upon the physical properties of the
fluid, but also the operating conditions. Therefore, to describe the intensity of shear in a
turbulent system such as an agitated fermenter, it is easier to estimate shear rate du/dy
instead of shear stress.1 Even though we use the shear rate as a measure of the shear
intensity, we should remember that it is the shear stress that ultimately affects the living
cells or enzyme. Depending upon the magnitude of viscosity and also whether the flow
is laminar or turbulent, there is a wide range of shear stress generated for the same
shear rate. However, the shear rate is a good measure for the intensity of the agitation.

Chapter seven
Fermentation Process Control
(Bioprocess control and Biosensor)

Introduction
 The growth of an organism in a bioreactor has to be controlled, so the operators must
have sufficient information about the state of the organism and the bioreactor
conditions.
 Monitoring a fermentation process may need basic knowledge of the bioprocess, and
the running conditions should be recorded.
There are direct and indirect measurements of microbial growth.
The methods of direct growth measurement are:
 cell optical density,
total cell counters,
Coulter counter,
cell dry weight,
packed cell volume and
optical detectors.
Growth is based on absorbance/light scattering. The absorbance of culture is generally
measured with spectrophotometer at a wavelength of about 600 nm.1

Cont…
The indirect measurements of cell growth are:
 based on cellular components,
measurements of ATP,
bioluminescence,
substrate consumption and
 product formation,
oxygen uptake rate,
respiration quotient and
heat evolution.

Instrumentation control for continuous stirred tank (CSTR) bioreactor.

Bioreactor operating parameters

BIOREACTOR CONTROLLING PROBES
 In bioprocess plant instrumentation and process control, variables are very
important.
 Reading and processing the information about the biosystem and monitoring the
cells are the major aims of the process instrumentation.
Controlling pH, measuring the dissolved oxygen in the fermentation broth and
controlling foam are all considered major parameters and necessary biological
information for large-scale operations.
 The main objective is to operate a bioreactor without any problems.
To do so we need to be familiar with all the controlling facilities and process
instrumentation.
Application of biosensors in the bioreactors is very common, so it is good to
know how the controlling unit operates.

CHARACTERISTICS OF BIOREACTOR SENSORS
 The sensor in a bioreactor provides knowledge and information on the state of
the process and also supplies suitable operational data for the process variables.
 Some of the physical and chemical effects on the bioreactor have to be
translated to electrical signals, which can be amplified and then displayed on a
monitor or recorder and used as an input signal for a controlling unit.
In practice, the response of most of the processes follows a sigmoidal S-shaped
curve.
A similar response would be obtained if a dissolved oxygen probe were
suddenly removed from a vessel with depleted oxygen levels, the probe
transferred to a vessel with water maintained with supplied air passed through
the aqueous phase, and the system agitated for sufficient oxygen transfer so that
it is saturated in the liquid phase.
Air bubbles may interfere with sensor signals, and false readings can mislead
the bioreactor operation.

TEMPERATURE MEASUREMENT AND CONTROL
Control of temperature for fermentation vessels is required because of the
narrow range of optimal temperature.
Most fermenters operate around 30–36 °C, but certain fermentation may require
control of the temperature in a range of 0.5 °C; this can easily be obtained in
large bioreactors.
 To maintain bioreactor temperature within the limited range, the system may
require regulation of heating and cooling by the control system.
 Heat is generated in the fermenter by dissipation of power, resulting in an
agitated system; heat is also generated by the exothermic biochemical reactions.
At the start and end of the fermentation, the heat generation rate is very low,
although the systems are normally heated to achieve the desired temperature.


Cont…
There are many alternatives for measuring and controlling the bioreactor
temperature. These include
 glass thermometers,
thermocouples,
 thermistors,
resistance thermometers
and miniature integrated circuit devices.
Thermal units capable of giving direct electrical output signals are favoured for
control purposes.
 Thermocouples are cheap and simple to use, but they are rather low in
resolution and require a cold junction.
 Thermistors are semiconductors, which exhibit a change in electrical
conductivity with temperature.

DO MEASUREMENT AND CONTROL
The dissolved oxygen content of the fermentation broth is also an important
fermentation parameter, affecting cell growth and product formation.
The rate of oxygen supply to the cell is often limited because the solubility of
oxygen in fermentation is low.
Unfortunately, measurement and control of dissolved oxygen under bioreactor
conditions is a challenging problem.
 The low solubility of oxygen makes the measurements very difficult.
There are several methods to determine the concentration of dissolved oxygen.
(1) the tubing method;
(2) use of mass spectrometer probes; and
(3) electrochemical detectors.

Cont…
 tubing method,
an inert gas flows through a coil of permeable silicon rubber tubing, which is
immersed in the bioreactor.
Oxygen diffuses from the broth, through the wall of the tubing and into a flow
of inert gas passing through the tube.
The concentration gradient exists because of the diffusion of oxygen into the
inert gas.
Then the concentration of oxygen gas in the inert gas is measured at the outlet
of the coil by an oxygen gas analyser.
This method has a relatively slow rate of response, of the order of several
minutes.
The advantages of this method are that it is simple and in situ sterilisation is
easily carried out.

Cont…
 use of mass spectrometer probes
 In the second method, the membrane of a mass spectrometer probe is used to
separate the fermenter contents from the high vacuum of the mass
spectrometer.
 Measurement of oxygen in the mass spectrometer probe and the tubing
method is based on the ability of the gas to diffuse across the surface
membrane.
 The most common method of measuring dissolved oxygen is based on
electrochemical detector.
 Two types of detector are commercially available: galvanic and polarographic
detectors.
 Both use membranes to separate electrochemical cell components from the
broth.
 The membrane must be permeable only to oxygen and not to any other
chemicals, which might interfere with the measurement.

Cont…
 Oxygen diffuses from the broth, across the permeable membrane to the
electrochemical cell of the detector, where it is reduced at the cathode to
produce a measurable current or voltage, which is proportional to the rate of
arrival of oxygen at the cathode.
 In the galvanic detector, the electrochemical detector consists of a noble metal
like silver (Ag) or platinum (Pt), and a base metal such as lead (Pb) or tin (Sn),
which acts as anode. The well-defined galvanic detector is immersed in the
electrolyte solution.
Various electrolyte solutions can be used, but commonly they may be a
buffered lead acetate, sodium acetate and acetic acid mixture.
 Polarographic electrodes are different from the galvanic type. In this type of
electrode the external negative base voltage is applied between the cathode (Au
or Pt) and the anode (Ag/ AgCl) so that oxygen is reduced at the cathode

pH/REDOX MEASUREMENT AND CONTROL
The fermentation process is normally carried out at a constant pH.
 The pH of a culture medium will change with the metabolic product of
microorganisms, which are developed in the fermentation media.
Therefore, pH control is required during the course of fermentation.
The pH has a major effect on cell growth and product formation by influencing
the breakdown of substrates and transport of both substrate and product through
the cell wall.
 In fine chemicals, organic acids, amino acids and antibiotic fermentations,
even a small change in the pH can cause a large fall in the productivity. Also, in
animal-cell fermentations, the pH is strictly affected by the cell density.
 Measurement of pH is based on the absolute standard of the electrochemical
properties of the standard hydrogen electrode.

DETECTION AND PREVENTION OF THE FOAM
 In a gas and liquid system, when gas is introduced into a culture medium,
bubbles are formed.
The bubbles rise rapidly through the medium and dispersion of the bubbles
occurs at surface, forming froth.
The froth collapses by coalescence, but in most cases the fermentation
broth is viscous so this coalescence may be reduced to form stable froth.
 Any compounds in the broth, such as proteins, that reduce the surface tension
may influence foam formation.
The stability of preventing bubbles coalescing depends on the film elasticity,
which is increased by the presence of peptides, proteins and soaps.
On the other hand, the presence of alcohols and fatty acids will make the foam
unstable.

Cont…
Foaming of the bioreactor is a nuisance, reflects on the mass transfer process and
must be prevented, for many reasons.
The problems related to foaming are obvious if they are due to gas sparging.
The problems are the loss of broth, clogging of the exhaust gas systemand
possible contamination, a problem that is due to wetting of the gas filters.
During fermentation, foaming may occur suddenly.
Some foams are easy to destroy and can be removed by foam breaker; others are
quite stable and are relatively hard to remove from the top of the bioreactor.

Cont….
The suppression of foam is usually accomplished by mechanical agitation such
as a foam breaker.
The mechanical devices operate on the centre of the shaft.
They are generally blades or disks that are mounted on the same agitator shaft.
Chemical anti-foams are used to regulate the foam and prevent any foaming on
the surface of broth.
The chemical anti-foams are expensive and minimal amounts must be used. Use
of chemical anti-foam may complicate the microbial fermentation process, and
some may act as an inhibitor.
Therefore they have to be regulated to eliminate any side effect on the
bioprocess. Chemical anti-foams are usually based on silicon and act by reducing
the interfacial tension of the broth.
 Mechanical devices have advantages because expensive chemicals do not have
to be added to the fermentation broth.

BIOSENSORS
The biochemical constituents of fermentation broth have been developed by a
wide range of biosensors.
A biosensor consists of two main elements.
These two elements, the biocatalyst and a transducer, are combined as a single
detecting probe, in which the transducer and biocatalyst are held together in a
very close contact.
The biosensor acts as a device as flow passes. It can detect penetration of flow
through biocatalysts and measures the biochemical transformation of a given
substance, for example change in pH.
The function of the transducer is to detect such change and to produce an output
signal, which is related to the concentration of the measured substance.
In fact, a biosensor is a combination of biological sensor attached to transducer
that is a simple device which acts specifically with a high sensitivity in
measurements.

Cont…
The application of biosensors in an operating bioreactor is usually based on
whole cells or enzyme activities.
The perfect function of a biosensor is very dependent on the biological
activities of a system.
The biocatalytic reaction produces some detectable change that must be
converted to an output signal by the transducer. The transducers are usuall
amperometric and potentiometric devices.
 Such transducers are included in dissolved oxygen probes and pH electrodes,
ion selective electrodes and gas-sensing devices.
Amperometric detectors operate by measuring the flux of som electrochemical
redox activities of the produced biosensor reaction.
For example, a dissolved oxygen probe can be used to measure the rate of
oxygen flux produced in an oxidised catalysed reaction.

Cont….
DO probes are a popular form of biosensor transducer. They are used for
microbial and enzymatic oxido-reductase reactions.
Many enzymatic reactions are associated with the uptake or production of
protons.
The rate of proton flux can be measured by using a potentiometric detector,
which is normally done in a pH probe.
Several biosensors are commercially available. One of the most useful is the
glucose sensor.
The standard sensor determines glucose concentration based on the glucose
oxidase enzyme.

Chapter eight
Downstream Processing

Introduction
After successful fermentation or enzyme reactions, desired products must be
separated and purified.
This final step is commonly known as downstream processing or bioseparation,
which can account for up to 60 percent of the total production costs, excluding
the cost of the purchased raw materials .
Fermentation products can be the cells them selves (biomass), components
with in the fermentation broth (extracellular) ,or those trappedin cells
(intracellular).
In the case of extracellular products, after the cells are separated, products in
the dilutea queous medium need to be recovered and purified.
The intracellular products can be released by rupturing the cells and then they
can be recovered and purified.

Major process steps in downstream processing

SOLID-LIQUID SEPARATION
The first step in downstream processing is the separation of solubles from the fermentation broth.
The selection of a separation technique depends on the characteristics of solids and the liquid
medium.
The product requires a sequence of operations for high purification.
The usual steps to follow are as follows.
1) Removal of insoluble particulates using various separation techniques:
filtration,
centrifugation and/or
settling/sedimentation/decanting.
(2) Primary isolation is done to increase product concentration:
Solvent extraction,
absorption,
precipitation and
ultrafiltration are the best known.
(3) Product purification. For production of highly pure product the impurities have to beremoved for
further product concentration such as
chromatography and
 adsorption

CONT…
(4) Final product isolation and drying of the crystallised product are done by
drum drying,
spray drying or
freeze drying.

FILTRATION
In filtration, solid particles are separated from a fluid–solid mixture by forcing the
fluid through a filter medium or filter cloth, which retains the solid particles.
As a result, solids are retained by filter media and the filtrate is obtained, which is a
clear solution without any solid particles.
The solid particles deposited on the filter form a layer, which is known as filter cake.
The deposited solids create resistance which reduces filter flux.
The depth of the filter cake gradually increases as more solids are retained.
 The filter cake may create more resistance to further filtration.
Filtration can be performed using either vacuum or positive-pressure equip-ment.
 The exerting differential pressure across the filter separates fluid from solid and is
called the filtration pressure drop.
 Ease of filtration depends on particle properties and fluid filtrates.
The compaction of particles, either soft or hard, compressible or non-compressible,and
the viscosity of the fluid may create different resistances.

Theory of Filtration
Assume laminar flow of filtrate of liquid through the filter cake.
 Rate of filtration is usually measured as the rate at which liquid filtrate is
collected.
The filtration rate depends on the area of the filter cloth, the viscosity of the
liquid, the pressure drop across the filter and filter cake resistance.
At any instant during filtration, the rate of filtration is given by the equation

CENTRIFUGATION
Centrifugation is used to separate materials of different densities when a force
greater than gravity has been implemented, such as centrifugal forces.
Centrifugation may be used to remove cells from fermentation broth; yeast, for
example, is harvested in a centrifuge unit.
For dilute suspensions each cell may be treated as a single particle in an infinite
fluid.
 In the concentrated fluid with suspended solids, the particle’s motion is
influenced by neigh-bouring particles.
A continuous process is commonly used in separation of solid parti-cles from
fermentation broth.
The particle’s velocity correlates with the hindered settling

Theory of Centrifugation
The particle’s velocity in a particular centrifuge is compared with the settling
velocity that occurs under the influence of gravity and the effectiveness of
centrifugation. The terminal velocity during gravity settling of a small particle
in dilute suspension is given by Stoke’s law:
 the centrifugal force is implemented to obtain terminal velocity in the
centrifuge:

SEDIMENTATION
When cells have a high tendency to aggregate closely (coagulate) or to form
multi cellular flocs with the aid of polyvalent cations or extracellular polymers,
the recovery of cell biomass becomes simple and easily applicable by the
sedimentation process.
Such aggregation pro-vides cell recycle streams in activate sludge wastewater
treatment; and several highly floccu-lent yeast strains are used in brewing beer
and single-cell protein production.
In fact, we need to remove cells from fermentation broth, so sedimentation is
considered as a downstream pro-cessing method.
Alum, lime and polyelectrolytes are commonly used to create macroflocs.
There are several natural and chemical coagulants used for aggregating
suspended cells in bioprocesses.

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