Coreconceptsofbiochemicalengineering1 150509053327 Lva1 App6891 PDF

Core Concepts of
Biochemical Engineering
Presented by: Raja Wajahat
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Introduction
Biotechnology
Biotechnology is the art and science of converting reactants into
useful products by the action of microorganisms or enzymes.
Examples:
production of a particular chemical, production of better plants/seeds,
use of specially designed organisms to degrade wastes
Bio-processing
Any process in which microorganisms play an essential role in getting
transformation of feed into useful products is called as bio-
processing.
Presented by Raja Wajahat

3
Biochemical Engineering is the extension of chemical engineering
principles to systems using a biological catalyst to bring about desired
chemical transformations.
It is usually divided into biochemical reaction engineering and bio
separations.
Biochemical Engineering is an important area in modern
biotechnology.

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Cells culture be scaled up, biological products be separated, purified
and prepared on a large scale.
Biochemical engineering is expected to carry out the above tasks and
to bring about huge economic benefits in realizing sustainable
development.

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It is the key to biotechnology development to intensify the researches
into biological reactors and the separation, purification technologies
for biological products.
And biochemical engineering has been playing an increasingly
important role in the above research fields.

6 Difference between bioprocess
and biochemical engineering
In addition to chemical engineering, bioprocess engineering would
include the work of mechanical, electrical and industrial engineers to
apply the principles of their disciplines to processes based on using
living cells.
Biologists and Engineers differ in their approach to research

In life sciences, mathematical theories and quantitative methods
(except statistics) have played a secondary role.
Results are qualitative and descriptive models are formulated and
tested.
However, biologists are very strong with respect to laboratory tools
and interpretation of laboratory data from complex systems.

Engineers possess good background in the physical and mathematical
sciences
Quantitative models and approaches even to complex systems are
strengths
The skills of engineer and life scientist are complimentary

9 Traditional and Modern Applications
of
Biotechnology/Bio-processing
Traditional
Foods, bakery products, beverages, wine from fruit juices,
fermentation of milk to make curd
Modern
Commercial production of antibiotics, vaccines, fermented foods,
organic acids etc.

Biochemistry
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What is Biochemistry?
Study of life cyclic processes in terms of chemicals
How life cycle proceeds with mutual cooperation of various activities
of living beings
Energy is released by breaking of the high energy storing molecules
usually phosphate containing molecules
Oxidation of NADH (nicotinamide adenine dinucleotide ) in the
mitochondria is one of the main reactions

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Biochemistry
Some of the chemical/biochemical reactions in the living organisms
are facilitated by another type of compounds called enzymes
Facilitation of a reaction is called as catalysis
Hence enzymes are called as biocatalysts or biological catalysts
Cells themselves contain some of the enzymes

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Biochemistry
Living organisms contain various bimolecules which are the building
blocks of the cell and also help in storing and releasing energy for
biotransformations
Living organisms contain a large number of bimolecules and
they are essentially composed of carbon and nitrogen. The
bimolecules have high molecular weights and are complex in
structure
They include carbohydrates, lipids, proteins, nucleic acids,
vitamins etc.

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Important Biomolecules
Carbohydrates
Lipids
Proteins
Nucleic acids

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Carbohydrates
Carbohydrates are made from monomers called monosaccharides.
Some of these monosaccharides include glucose (C6H12O6),
fructose (C6H12O6), and deoxyribose (C5H10O4).
When two monosaccharides undergo dehydration synthesis, water is
produced, as two hydrogen atoms and one oxygen atom are lost from
the two monosaccharides' hydroxyl group.

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Carbohydrates

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Carbohydrates

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LIPIDS
Lipids are usually made from one molecule of glycerol combined
with other molecules.
In triglycerides, the main group of bulk lipids, there is one molecule
of glycerol and three fatty acids.
Fatty acids are considered the monomer in that case, and may be
saturated (no double bonds in the carbon chain) or unsaturated (one or
more double bonds in the carbon chain).

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LIPIDS
Lipids, especially phospholipids, are also used in various
pharmaceutical products,
either as co-solubilisers (e.g., in parenteral infusions) or
else as drug carrier components (e.g., in a liposome or transfersome).

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LIPIDS

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LIPIDS
Class of compounds which are fatty/oily in nature and present in cells
and tissues
In addition to fats and oils, some other biological materials including
waxes, cholesterol and some vitamins and hormones are also
classified as lipids.
General structure of fats and oils
Triglycerides are formed due to the reaction of alcohol glycerol and
long chain fatty acids such as stearic acid

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Lipid Structure

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Characteristics of Lipids
Insoluble in water
Soluble in non-polar solvents including hexane, chloroform etc
Release a lot of energy on breakdown and therefore considered as the
energy storage media
Contain a large proportion of C-H bonds
Upon saponification, release fatty acids and glycerol
They are synthesized by the cells from sugars
Some lipid compounds such as vitamins and hormones have intense
biological activity

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As bimolecules, they are constituted of cells wall and form a
protective coating to the cell and encourage some species.
They are also energy carriers and release energy as and when cell
requires it

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Lipids also include a heterogeneous group of structural component.
Some lipids are combined with other classes of compounds and they
are known as:
Lipoproteins,
Proteolipids,
Lipoamino acids,
Phosphatidopeptides,
Lipopolysaccharides

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Proteins
Proteins are very large molecules macro-biopolymers made from
monomers called amino acids.
There are 20 standard amino acids, each containing a carboxyl group,
an amino group, and a side-chain (known as an "R" group).
The "R" group is what makes each amino acid different, and the
properties of the side-chains greatly influence the overall three-
dimensional conformation of a protein.
When amino acids combine, they form a special bond called a peptide
bond through dehydration synthesis, and become a polypeptide, or
protein.
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Proteins

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Proteins

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Proteins
In order to determine whether two proteins are related, or in other
words to decide whether they are homologous or not, scientists use
sequence-comparison methods.
Methods like Sequence Alignments and Structural Alignments are
powerful tools that help scientists identify homologies between
related molecules.
The relevance of finding homologies among proteins goes beyond
forming an evolutionary pattern of protein families.
By finding how similar two protein sequences are, we acquire
knowledge about their structure and therefore their function.
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Nucleic acids
Nucleic acids are the molecules that make up DNA, an extremely
important substance that all cellular organisms use to store their
genetic information.
The most common nucleic acids are deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA).
Their monomers are called nucleotides.
A nucleotide consists of a phosphate group, a ribose sugar, and a
nitrogenous base.

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Nucleic acids
The phosphate group and the sugar of each nucleotide bond with each
other to form the backbone of the nucleic acid, while the sequence of
nitrogenous bases stores the information.
The most common nitrogenous bases are adenine, cytosine, guanine,
thymine, and uracil.

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Nucleic acids
The nitrogenous bases of each strand of a nucleic acid will form
hydrogen bonds with certain other nitrogenous bases in a
complementary strand of nucleic acid (similar to a zipper).
Adenine binds with thymine and uracil; Thymine binds only with
adenine; and cytosine and guanine can bind only with one another.

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Nucleic acids

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GENERALIZED VIEW OF BIOPROCESS
RAW MATERIALS
UPSTREAM PROCESSES
Media
Inoculum Equipment Formulation
Preparation Sterilization and
Sterilization
BIOREACTOR - FERMENTER
Reaction
Instrumenta
Kinetics Transport Phenomena
tion
and and Fluid Properties
and Control
Bioactivity
DOWNSTREAM PROCESSES
Waste
Recovery and Recovery,
Separation
Purification Reuse and
Treatment
THE BOTTOM LINE
HEALTH
REGULATIO ECONOMIC
AND
Presented by Raja Wajahat N S
SAFETY
Microbiology
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Microbiology
Microbiology is the study of microscopic organisms, those being
unicellular (single cell), multicellular (cell colony), or acellular
(lacking cells).
Microbiology encompasses numerous sub-disciplines including
virology, mycology, parasitology, and bacteriology.

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Microbiology
Eukaryotic micro-organisms possess membrane-bound cell organelles
and include fungi and protists, whereas prokaryotic organisms
which all are microorganismsare conventionally classified as
lacking membrane-bound organelles and include eubacteria and
archaebacteria.
Microbiologists traditionally relied on culture, staining, and
microscopy.
However, less than 1% of the microorganisms present in common
environments can be cultured in isolation using current means
Microbiologists often rely on extraction or detection of nucleic acid,
either DNA or RNA sequences.
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Microbiology
Viruses have been variably classified as organisms, as they have been
considered either as very simple microorganisms or very complex
molecules.
Prions, never considered microorganisms, have been investigated by
virologists, however, as the clinical effects traced to them were
originally presumed due to chronic viral infections, and virologists
took searchdiscovering "infectious proteins".

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Microbiology
As an application of microbiology, medical microbiology is often
introduced with medical principles of immunology as microbiology
and immunology.
Otherwise, microbiology, virology, and immunology as basic sciences
have greatly exceeded the medical variants, applied sciences

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Microbiology
Study of microscopic organisms
Important branch of science
As a basic biological science
Deals with nature of life processes and principles behind,
genetics
As an applied biological science
Study of useful as well as pathogenic microorganisms

41 Why microbiology is
important?
In biochemical engineering
To understand and analyze the process of biotechnology
Design and operate different units in rational a way
Therefore, a basic knowledge of cell growth and function is
required
A living microorganism may be conceptualized as a chemical
reactor (take nutrients from environment, grows, reproduces and
releases products)
Products formed and released during cellular activities could be
commercially important

42 Why microbiology is
important?
Rates of nutrient utilization, growth and release of products depends
upon:
Type of the cells involved
Temperature
Composition of media etc.
Quantitative understanding of biological systems (correlation of
friction factor and Reynolds No.)
Understanding above interactions requires a foundation built on
microbiology and biochemistry
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Industrial Microbiology
Study of the exploitation of the biochemical potential of microbes for
the production of various products
Antibiotics, vaccines, steroids, solvents, vitamins etc.
Developments of new products using genetic engineering

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What Are Microorganisms?
Microorganisms are actually a diverse group of organisms.
The fact that theyre micro isnt even true of all microorganisms
some of them form multicellular structures that are easily seen with
the naked eye

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There are four main kinds of microorganisms, based on evolutionary
lines:
Bacteria are a large group of unicellular organisms that scientists
loosely group as Gram-negative and Gram-positive, but in reality
there are many different kinds.
The bacteria and archaea are often talked about together under the
heading of prokaryotes because they lack a nucleus. They do share
a few characteristics and arent easily distinguished from one another
at first, but they are distinct groups.

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Archaea are another group of unicellular organisms that evolved
along with bacteria several billion years ago.
Many are extremophiles, meaning that they thrive in very hot or very
acidic conditions.
Archaea are more closely related to eukaryotes than to bacteria.

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Eukaryotic microorganisms are a structurally diverse group that
includes protists, algae, and fungi.
They all have a nucleus and membrane-bound organelles, as well as
other key differences from bacteria and archaea.
All the rest of the multicellular organisms on earth, including humans,
have eukaryotic cells as well.

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Viruses are smaller than bacteria and are not technically alive on their
own they must infect a host cell to survive.
Viruses are made up of some genetic material surrounded by a viral
coat, but they lack all the machinery necessary to make proteins and
catalyze reactions.
This group also includes subviral particles and prions, which are the
simplest of life forms, made of naked ribonucleic acid (RNA) or
simply protein.

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Genetic Engineering

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Microscopy
Microorganisms are measured in smaller units such as microns,
nanometers, mill microns and Angstrom
Various microscopes
Difference between ordinary and electron microscope

52 Range of microscopic
measurements

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Building block of organisms
All living organisms are composed of cells

What is true for Escherichia coli is true for elephants
Cells are b/w 1 and 50 micrometer in diameter
Basic components of living cell
Cytoplasm
Cell membrane
Nucleus
Ribosome
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Cell Nucleus (DNA Structure)

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Cell components

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DNA
DNA determines When DNA is damaged by
Heredity foreign substances, various
toxic effects, including:
Cell reproduction
Mutations
Protein synthesis
Cancer
Birth effects
Defective immune system

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Cell Membrane
Acts as a barrier from external environment
It closes the cell and regulates the passage of ions, nutrients,
metabolic products and fat soluble substances into and out of it
It is composed of phospholipid bilayer about 8 nm thick
Highly selective membrane enabling the cell to concentrate specific
metabolites and excrete waste
A number of complex transformation takes place across the
membrane

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Cytoplasm

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Cytoplasm
Colloidal in nature
Thick semi-transparent and has higher water contents
It contains:
Hydrophilic components (protein particles,
carbohydrates and salts)
Hydrophobic components (lipids or fats)
Main function of cytoplasm is absorption and excretion

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Prokaryotes and Eukaryotes
Prokaryotic cell Eukaryotic cell

Genetic material is not Eukaryote means true
enclosed within the nucleus
membrane
Genetic material enclosed
Cell walls contain complex in a specialized membrane
polysaccharide
They are larger and more
peptidoglycan
complex than prokaryotes
Simple method of
Size range from 2 to 200
reproduction
micrometer
Size is usually 0.5 to 3
micrometer
Presented by Raja Wajahat in diameter
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Applications of Prokaryotes
Metabolically the most diverse of all living systems

Responsible for most degradation processes
Can be grown aerobically and anaerobically
Form a wide range of organic products (this property has both
positive and negative impact on society)

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Applications of Prokaryotes
Positive
represent a massive resource of biocatalysis for the
biotransformation of organic materials and the degradation of
herbicides, insecticides and other man-made chemicals
Negative
Represent the principal agents causing the deterioration of
biomaterial e.g food and wood and are major hazards to public
health (food poisoning and other diseases)

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Classification of organisms
Classified according to their structure and function

Divided into three kingdoms
Plants
Animals
Protists (Neither plants nor animals)
Most are unicellular but some have many cells
Cells have a membrane around the nucleus (eukaryotes)

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Classifications show differences in several characteristics including:

Energy and nutritional requirements
Rates of growth and product release
Method of reproduction
Morphology

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Naming the microorganisms
They are named in Latin using binary nomenclature

First name represents the group or genus
Second name represents the species
Escherichia coli C600
National collection of industrial and marine bacteria (NCIMB)
American type culture collection (ATCC)
Strain (A strain is a subset of a bacterial species differing from
other bacteria of the same species by some minor but identifiable
difference)

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Escherichia coli (E. coli)
Escherichia coli (E. coli) chosen as a test microorganism.
E. coli is currently the most specific indicator for faecal
contamination of a water source and therefore it is considered as a
model organism in laboratory research.
The cells are about 2 m long and 0.5 m in diameter, with a cell
volume of 0.6 0.7 m3 (Kubitschek, 1990).

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Optimal growth of E. coli occurs at 37C. Under a microscope,
E. coli is a rod-shaped prokaryotic cell which has a long, rapidly
rotating flagellum (tail) used for movement.
A strain of E. coli is a sub-group within the species that has unique
characteristics that distinguish it from other E. coli strains.

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These differences are often detectable on the molecular level and may
result in changes to the physiology or life cycle of the bacterium.
For example, a strain may gain pathogenic capacity or the ability to
resist antimicrobial agents.
Different strains of E. coli are often host-specific, making it possible
to determine the source of faecal contamination in environmental
samples.

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Different Bacteria
Pseudomonas aeruginosa (P. aeruginosa)
is a gram-negative rod shaped free living bacterium that is ubiquitous
in the environment
Staphylococcus aureus (S. aureus)
is a gram positive bacterium usually arranged in grape like irregular
clusters.
Although it occurs widely in the environment it is found mainly on
skin and the mucous membranes of animals.

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Different Bacteria
S. aureus can be released into environments including swimming
pools, spa pools and other recreational waters by human contact.
Legionella pneumophila (L. pneumophila)
is a gram negative rod shaped bacterium.

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Yeasts
Rhodosporidium turoloides (R. turoloides)
Y4 is oil producing or oleaginous yeast (Wu et al. 2011).
Since these species contain intracellular valuable compounds such as
lipids, therefore the disruption of this yeast would be interesting in
order to release the lipids contained in vacoules within the yeast cell.
Once the lipids are released biodiesel could be produced via a
conventional trans-esterification process.

Enzymes
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What are Enzymes?
Enzymes are biological catalysts and are one of the essential
components of all living systems
Biochemical reactions occur rapidly through the mediation of natural
catalysts called enzymes
Enzymes are bimolecules that catalyze (increase the rates of)
chemical reactions
Enzymes have a key role in catalysing the chemical transformations
that occur in all cell metabolism without themselves undergoing any
overall change

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Enzymes
Some generic terms associated with enzymology:
Cofactor: the non-protein content of enzyme
Coenzyme: an enzyme with organic molecules as its cofactor
Haloenzyme: an active enzyme including cofactor
Apoenzyme: the inactive portion of protein
The nature and specificity of their catalytic activity is basically due to
the three dimensional structure of folded protein (determined by the
sequence of amino acids)

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Classification of Enzymes
Enzymes are usually named in terms of the reactions that are
catalysed
Usual practice is to add ase to the major part of the name of the
substrate e.g Urease, Urginase (urginine)
Enzymes are also classified by groups that catalyse similar reactions
(see slide 17)

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Properties of enzymes
The catalytic activity of enzymes differs from that of other catalysts
Efficiency
Turn over number= molecules reacted per catalytic site per unit
time
Turn over number for enzymes at room temperature are usually
much higher than for industrial chemical catalysts

79
Specificity of enzymes
Specificity
A characteristic feature of enzymes is that they are specific in
action, some showing complete specificity for only one type of
molecule
If a substance exists in two stereochemical forms, L and D isomers,
enzymes may recognize only one of the two forms for example
glucose oxidase will oxidise D(+) glucose only and no other hexose
isomer.

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Specificity of enzymes
Active centre/Active site
A catalyst site on the molecule is called active site/active centre.
Such sites constitute only a small proportion of the total volume
of the enzyme and are located on or near the surface.
The active site is usually a very complex physico-chemical
space, creating microenvironments in which the binding and
catalytic areas can be found.
The forces operating at the active centre can involve
Charge, hydrophobicity, hyfrogen bonding and redox
processes
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How the biological catalysts work?
A reaction proceeds according to the two possible theories

Collision theory
Proposes that reactions take place by the collision of the
reactant molecules. More is the concentration of the
reactants, more are the chances for the reactants to collide
and hence more will be the rate of reaction. However, all
collisions may not necessarily result in the reaction to
proceed to produce products

82
How the biological catalysts work?
Transition state theory

Propose that the collision of certain molecules which have
crossed certain potential energy barrier alone will result in
the reaction to take place. This potential energy barrier is
known as activation energy

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Biological catalysts
Like all catalysts, enzymes work by lowering the activation energy
for a reaction thus increasing the reaction rate
Not consumed by the reaction
Do not alter the equilibrium
Enzymes differ from most other catalysts by being much more
specific
Enzymes are know to catalyze about 4000 biochemical reactions

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Types of specificity
Depending upon the reaction conditions and the specific nature of
t5he enzymes, the enzymatic catalytic process exhibits different kinds
of specificity including;
Group specificity
Stereochemical specificity
Product specificity
Substrate specificity

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Enzymatic process

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Enzyme specificity hypothesis
Several hypothesis have been proposed to explain the enzyme

specificity in catalytic activity and its ability to interact with the
substrates
Fischer lock and key hypothesis
It was proposed by Fischer in 1890 who conceived the
concept of complementary structural features between the
enzyme and the substrate

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The catalytic process is brought about because the substrate

fits into the complementary site on the enzyme just as key
fits into the lock
Thus, the reacting group of the substrate gets struck with the
catalytic site of the enzyme
Similarly, the binding groups attach to the binding sites in
the enzyme
Hypothesis has been successful in explaining many features
of the enzyme specificty

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Drawback
Could not explain some of the conformational changes taking
place in the enzymes when they come in contact with the
substrate
An enzyme may not be having exactly complementary feature
that is compatible to the substrate, but still there are cases where
reaction have taken place

90
Drawback
X-ray diffraction analysis and some spectroscopic analysis have
shown differences in the structures of free enzymes and substrate
bound enzymes.
This was explained by Koshland in 1958 with his Koshland
induced-fit hypothesis

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Koshland induced-fit hypothesis

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Koshland induced-fit hypothesis
This hypothesis proposes that the structure of the substrate may not
be complementary to the enzyme in its native format,
but it is complementary to the active site in the substrate-enzyme
complex.
Both the enzyme and the substrate change their structure slightly to
accommodate each other.

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Versatility
Enzymes catalysis is shown by the type of reactions that can be catalysed. Six
groups of enzymes are recognized according to their reactivity
1. Oxidoreductase.oxidation-reduction reactions
2. Transferases..transfer of atom b/w two molecules
3. Hydrolases..hydrolysis reactions
4. Lyases.removal of a group from a substrate
5. Isomerases..isomerisation reactions
6. Ligasescatalyse the synthesis of various types of bonds
where the reactions are coupled with breakdown of energy-containing
materials such as ATP

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Difference b/w catalyst and enzyme
Function:
Catalysts are substances that increase or decrease the rate of a
chemical reaction but remain unchanged.
Enzymes are proteins that increase rate of chemical reactions
converting substrate into product.
Molecular weight:
Low molecular weight compounds.
High molecular weight globular proteins.

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Types:
There are two types of catalysts positive and negative catalysts.
There are two types of enzymes - activation enzymes and inhibitory enzymes.
Alternate terms:
Inorganic catalyst. Organic catalyst or bio catalyst.
Nature:
Catalysts are simple inorganic molecules
Enzymes are complex proteins

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Reaction rates:
Typically slower Several times faster
Specificity:
They are not specific and therefore end up producing residues with
errors Enzymes are highly specific producing large amount of good
residues
Conditions:
High temp, pressure
Mild conditions,

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Enzymes are proteins, which act as catalysts.

Enzymes lower the energy required for a reaction to occur, without
being used up in the reaction.
Many types of industries, to aid in the generation of their products,
utilize enzymes.
Examples of these products are; cheese, alcohol and bread.

98
Fermentation
Fermentation is a method of generating enzymes for industrial

purposes.
Fermentation involves the use of micro organisms, like bacteria and
yeast to produce the enzymes.
There are two methods of fermentation used to produce enzymes.

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Fermentation
These are submerged fermentation and solid-state fermentation.

Submerged fermentation involves the production of enzymes by
microorganisms in a liquid nutrient media.
Solid-state fermentation is the cultivation of microorganisms, and
hence enzymes on a solid substrate.

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Enzymes
Carbon containing compounds in or on the substrate are broken down
by the micro organisms, which produce the enzymes either
intracellular or extracellular.
The enzymes are recovered by methods such as centrifugation, for
extracellular produced enzymes and lysing of cells for intracellular
enzymes.

101
Enzymes
Many industries are dependent on enzymes for the production of their
goods.
Industries that use enzymes generated by fermentation are the
brewing, wine making, baking and cheese making

Immobilization of Enzymes
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Immobilized Enzymes
The remarkable catalytic properties of enzymes make them very
attractive for use in processes where mild chemical conditions and
high specificity are required.
Cheese manufacture has traditionally used rennet, an enzyme
preparation from calf stomach, as a specific protease which leads to
the precipitation of protein from milk.

104
Immobilized Enzymes
Mashing in the malting of grain for the brewing of beer makes use
of pamylase from germinating grain to hydrolyse starch to produce
sugars for the fermentation
stage. In both of these examples the enzymes are not recovered from
the reaction mixture and a fresh preparation is used for each batch.

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Immobilized Enzymes
Similarly, in more modern enzyme reaction applications, such as in
biological washing detergents, the enzyme is discarded after single
use but there are, however, situations where it may be desirable to
recover the enzyme.
This may be because the product is required in a pure state or that the
cost of the enzyme preparation is such that single use would be
uneconomic.

106
Immobilized Enzymes
To this end, immobilized biocatalysts have been developed where the
original soluble enzyme has been modified to produce an insoluble
material which can be easily recovered from the reaction mixture.
Many industrially important micro-organisms tend to agglomerate
during their growth and form flocs suspended in the culture medium
or films which adhere to the internal surfaces of the fermenter.

107
Immobilized Enzymes
This tendency may or may not be advantageous to the process and is
dependent on a variety of parameters such as the pH and ionic
strength of the medium and the shear rate experienced in the growth
vessel.
In some cases the formation of substantial flocs is essential to the
proper operation of the process.

108
Immobilized Enzymes
In the case of the activated sludge waste water treatment the settling
properties of the flocculated micro-organisms are utilized in order to
produce a concentrated stream of biomass for the recycle.
The so-called trickling filter, also in widespread use in waste-water
treatment, is reliant on the formation of a film of organisms on the
surfaces of its packing material.
The operation is not that of a filter, in which material would be
removed on the basis of its particle size, but that of a biological
reactor in which the waste material forms the substrate for the growth
of the microbes.

109
Immobilized Enzymes
The presence of the film provides a means of retaining a higher
microbial concentration in the reactor than would be retained in a
comparable stirred-tank fermenter.
The formation of flom and films for the retention of high microbial
densities or to facilitate separation of microbes from the growth
medium may be desirable in other instances as well.
However, in some cases the microbe used may neither be amenable to
the natural formation of large flocs nor adhere as surface films, and
recourse may be made to the artificial immobilization of microbes.

110
Immobilization techniques
There are various methods which have been developed for enzyme
and microorganism immobilization and some of these have found
commercial application.
The two largest scale industrial processes utilizing immobilized
enzymes are the hydrolysis of benzyl penicillin by penicillin acylase
and the isomerisation of glucose to a glucose-fructose mixture by
immobilized glucose isomerase.
The immobilization techniques used in general may be broadly
categorized as:

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(a) Physical adsorption on to an inert carrier.
The first of these methods has the advantage of requiring only mild
chemical conditions so that enzyme deactivation during the
immobilization stage is minimized.
The natural formation of microbial flocs and films may be
considered to be in this category, although the subsequent adhesion
of the microbes to the surface may not be a simple phenomenon.
Special materials may be used as supports which provide the
microbes with environments which are particularly amenable to
their adhesion;
112
such materials include foam plastics which provide conditions of low
shear in their pores.
The process may also be relatively cheap but it does tend to have the
drawback that desorption of the enzyme may also occur readily or
that the microbial film may slough and be carried into the bulk of the
growth medium.
The process is dependent on the nature of the specific enzyme or
microbe used and its interaction with the carrier and, whilst it is
common in the case of immobilized microbes, it has found only
limited application in the case of immobilized enzymes.

113
(b) Inclusion in the lattices of a polymer gel or in micro-capsules.
This method attempts to overcome the problem of leakage by
enclosing the relatively large enzyme molecules or microbes in a
tangle of polymer gel or to enclose them in a membrane which is
porous to the substrate.
It is theoretically possible to immobilize any enzyme or micro-
organism using these methods but they too have their problems. Some
leakage of the entrapped species may still occur, although this tends
to be minimal.

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The main problem is due to mass transfer limitations to the
introduction of the necessarily small substrate molecules into the
immobilized structure, and to the slow outward diffusion of the
product of the reaction.
If the substrate is itself a macro-molecule, such as a protein or a
polysaccharide, then it will be effectively screened from the enzyme
or microbes and little or no reaction will take place.

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(c) Covalent binding
Biological catalysts may be made insoluble and hence immobilized
by effectively increasing their size.
This can be done either by chemically attaching them to otherwise
inert carrier materials or by cross linking the individuals to form large
agglomerations of enzyme molecules or micro-organisms.
The chemical reagents used for the linking process are usually
bifunctional, such as the carbo-di-imides, and many have been
developed from those used in the chemical synthesis of peptides and
proteins.

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The inert carriers used tend to be hydrophilic materials, such as
cellulose and its derivatives,
but in some cases the debris of the original cells has been used, the
cells having been broken and then crosslinked with the enzyme and
each other to form large particles.

117

118
The consumption or biotransformation of substrate by immobilized
micro-organisms results in most cases in the growth of the micro-
organisms.
The growth which gives rise to a significant increase of thickness in
an established biofilm, occurs at a rate which is essentially slow in
comparison with the rates of the diffusion processes.

119
Simultaneously, the attrition of biofilms or flocs arising from the
effects of fluid flow tends to maintain their thickness or size, and,
overall, the immobilized system can be considered to be in a steady
state when short time intervals are involved.
The mathematical similarity of enzyme and microbial kinetics then
means that a common set of equations can be used to describe the
behavior of both immobilized enzymes and microbial cells.

Thank You!
120
Presented by: Raja Wajahat

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Core Concepts of

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

All living organisms are composed of cells

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Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Presented by Raja Wajahat

Prokaryotic cell Eukaryotic cell

Metabolically the most diverse of all living systems

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Presented by Raja Wajahat

Classified according to their structure and function

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Classifications show differences in several characteristics including:

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Presented by Raja Wajahat