Core Concepts of Biochemical Engineering

Core Concepts of Biochemical Engineering

Presented by: Raja Wajahat

Introduction

Biotechnology

Biotechnology is the art and science of converting reactants intouseful 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.

2

Presented by Raja Wajahat

Biochemical Engineering

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.

3



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.

4



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.

5


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

6




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.

7




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

8


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.

9


Biochemistry


10

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

11


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

12


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.

13


Important Biomolecules

Carbohydrates

Lipids

Proteins

Nucleic acids


14

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.


15

Carbohydrates


16

Carbohydrates


17

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).

18


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).

19


LIPIDS


20

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

21


Lipid Structure


22

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

23



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

24



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

25


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.Presented by Raja Wajahat

26

Proteins


27

Proteins


28

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.Presented by Raja Wajahat

29

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.


30

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.


31

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.


32

Nucleic acids


33

GENERALIZED VIEW OF BIOPROCESSRAW MATERIALS

UPSTREAM PROCESSES

Inoculum

Preparation

Equipment

Sterilization

BIOREACTOR - FERMENTER

Reaction Kinetics

and

Bioactivity

Transport Phenomena

and Fluid Properties

DOWNSTREAM PROCESSES

SeparationRecovery and

Purification

THE BOTTOM LINE

REGULATIO

N

ECONOMIC

S

HEALTH AND

SAFETY

Waste Recovery,Reuse and

Treatment

Instrumentation

and Control

Media Formulation

and

Sterilization

34


Microbiology


35

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.

36


Microbiology

Eukaryotic micro-organisms possess membrane-bound cell organelles

and include fungi and protists, whereas prokaryotic organisms—

which all are microorganisms—are 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.

37


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 search—discovering "infectious proteins".

38


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

39


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

40


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

41


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

42


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

43


What Are Microorganisms?

Microorganisms are actually a diverse group of organisms.

The fact that they’re micro isn’t even true of all microorganisms

some of them form multicellular structures that are easily seen with

the naked eye


44


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 aren’t easily distinguished from one another

at first, but they are distinct groups.


45


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.


46


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.


47



48


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.


49

Genetic Engineering50


Microscopy

Microorganisms are measured in smaller units such as microns,

nanometers, mill microns and Angstrom

Various microscopes

Difference between ordinary and electron microscope

51


Range of microscopic

measurements

52


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

53


Cell Nucleus (DNA Structure)54


Cell components55


DNA

DNA determines

Heredity

Cell reproduction

Protein synthesis

When DNA is damaged by

foreign substances, various

toxic effects, including:

Mutations

Cancer

Birth effects

Defective immune system

56


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

57


Cytoplasm58


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

59


Prokaryotes and Eukaryotes

Prokaryotic cell

Genetic material is not

enclosed within the

membrane

Cell walls contain complex

polysaccharide

peptidoglycan

Simple method of

reproduction

Size is usually 0.5 to 3

micrometer in diameter

Eukaryotic cell

Eukaryote means true

nucleus

Genetic material enclosed

in a specialized membrane

They are larger and more

complex than prokaryotes

Size range from 2 to 200

micrometer

60


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)

61


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)

62


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)

63


Classification of organisms

Classifications show differences in several characteristics including:

Energy and nutritional requirements

Rates of growth and product release

Method of reproduction

Morphology

64


Classification of organisms65


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)

66


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 μm long and 0.5 μm in diameter, with a cell

volume of 0.6 – 0.7 μm3 (Kubitschek, 1990).


67



68


Optimal growth of E. coli occurs at 37ºC. 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.

69



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.

70


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.

71


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.

72


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.

73


Enzymes


74

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

75


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)

76


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)

77


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

78


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.

79


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

80


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

81


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

82


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

83


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

84


Enzymatic process 85


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

86



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

87


Fischer lock and key hypothesis88



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

89



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

90


Koshland induced-fit hypothesis91


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.

92


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. Ligases……………catalyse the synthesis of various types of bonds where the reactions are coupled with breakdown of energy-containing materials such as ATP

93


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.

94



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

95



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,

96



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.

97


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.

98


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.

99


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.

100


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

101


Immobilization of Enzymes


102

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.

103


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.

104


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.

105


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.

106


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.

107


Immobilized Enzymes

In the case of the activated sludge waste water treatment the settlingproperties of the flocculated micro-organisms are utilized in order toproduce a concentrated stream of biomass for the recycle.

The so-called ‘trickling filter’, also in widespread use in waste-watertreatment, is reliant on the formation of a film of organisms on thesurfaces of its packing material.

The operation is not that of a filter, in which material would beremoved on the basis of its particle size, but that of a biologicalreactor in which the waste material forms the substrate for the growthof the microbes.

108


Immobilized Enzymes

The presence of the film provides a means of retaining a highermicrobial concentration in the reactor than would be retained in acomparable stirred-tank fermenter.

The formation of flom and films for the retention of high microbialdensities or to facilitate separation of microbes from the growthmedium may be desirable in other instances as well.

However, in some cases the microbe used may neither be amenable tothe natural formation of large flocs nor adhere as surface films, andrecourse may be made to the artificial immobilization of microbes.

109


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:

110



(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;

111



such materials include foam plastics which provide conditions of lowshear in their pores.

The process may also be relatively cheap but it does tend to have thedrawback that desorption of the enzyme may also occur readily orthat the microbial film may slough and be carried into the bulk of thegrowth medium.

The process is dependent on the nature of the specific enzyme ormicrobe used and its interaction with the carrier and, whilst it iscommon in the case of immobilized microbes, it has found onlylimited application in the case of immobilized enzymes.

112



(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.

113



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.

114



(c) Covalent binding

Biological catalysts may be made insoluble and hence immobilizedby effectively increasing their size.

This can be done either by chemically attaching them to otherwiseinert carrier materials or by cross linking the individuals to form largeagglomerations of enzyme molecules or micro-organisms.

The chemical reagents used for the linking process are usuallybifunctional, such as the carbo-di-imides, and many have beendeveloped from those used in the chemical synthesis of peptides andproteins.

115



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.

116



117


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.

118



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.

119


Thank You!

Presented by: Raja Wajahat


120