its strain

1. BIOCHEMICAL ENGINEERING
 FTRI 1203 Biochemical Engineering
 Biological materials including mutation and gene cloning. Micro
organisms: energy yielding compounds, systems, accumulation of
metabolites, kinetic patterns of various fermentations. Kinetics:
enzyme systems, absolute reaction rate theory, steady state
continuous cultivation theory, microbial dynamics in chemostat
culture, batch and continuous cultivation with examples.
Aeration and agitation: mass transfer and microbial respiration,
bubble aeration and mechanical agitation, factors influencing
oxygen transfer coefficients. Media sterilization: batch and
continuous, air sterilization, design example of a filter for air
sterilization, PVA filter for air sterilization. Equipment design
and asepsis: fermenter design, cardinal rules, materials of
construction and vessel size, bearing assemblies, motor drive,
aseptic seals, aseptic operation, tangential flow filtration (TFF),
piping and valves for biochemical engineering, pressure relief,
cleaning and sterilization of process equipment
 Credit: 2

2. BIOCHEMICAL ENGINEERING
Biochemical Engineering
 Biological materials including mutation and gene
cloning.
 Micro-organisms: energy yielding compounds, systems,
accumulation of metabolites
 Kinetic patterns of various fermentations.
 Kinetics: enzyme systems, absolute reaction rate theory

3. BIOCHEMICAL
ENGINEERING
Application of scientific
and engineering
principles
processing of materials by biological
agents to provide new products and
services
Pharmaceutical, biotechnological and water
treatment industries
Chemical Engineering, Microbiology and
Biochemistry
It’s roots came out from brewing & leavening,
cheese, enzymes, tempeh, tofu, idli, porridge
Modern processes connected to Antibiotics
Streptomycin,
erythromycin,
tetramycin
and Vitamin
B12

4. CONCERN OF BIOCHEMICAL
ENGINEERING
Components of a typical Microbial Processes

5. BIOLOGICAL MATERIALS IN
BIOCHEMICAL PROCESSES
 Production/Industrial MOs
 Main tools for MP
 Traditional microbial processes >1000 years
 Pasteur 1857
 Hansen 1883; pure strains
 Strains for MP possessed special characteristics

6. They should ideally exhibit:
1. genetic stability;
2. efficient production of the target product
3. limited or no need for vitamins and additional growth factors;
4. utilization of a wide range of low-cost and readily available carbon sources;
5. amenability to genetic manipulation;
6. safety, non-pathogenicity and should not produce toxic agents, unless this
is the target product;
7. ready harvesting from the fermentation;
8. ready breakage, if the target product is intracellular; and
9. production of limited by-products to ease subsequent purification
problems.
PRODUCTION/ INDUSTRIAL
MICROORGANISMS

7. SOURCES MICROORGANISMS
Sources of IM
 Either isolated from environment
 Collected from culture collection

8. COLLECTION OF INDUSTRIAL
MICROORGANISMS
1. Isolation from Environment
 Two types of strategies adopted:
 Shotgun
 Free living MOs from man-made or natural habitants
 Objective approach
 By sampling from a specific site

9. 2. Culture Collection
 The main functions of culture collection are
 to maintain the existing collection
 to continue to collect new strains
 to provide pure and authenticated organisms
 Rich source MOs for past, present and future
 500 CC around the world
 UK National Culture Collection (UKNCC)
 American Type Culture Collection (ATCC)
 DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen)
COLLECTION OF INDUSTRIAL
MICROORGANISMS

10. IMPROVEMENT OF INDUSTRIAL
MICROORGANISMS
 Individual strain may not as efficient as it is
expected
 Irrespective of their source, they might need
Improvement
 Improvement is also related to regulatory
consideration; GRAS
 Mutation and cloning are concerned to improvement

11. IMPROVEMENT OF INDUSTRIAL
MICROORGANISMS
 Target of strain improvement
 Rapid growth
 Genetic stability
 Non-toxicity to human
 Large cell size; Convenient DSP
 Ability to use cheaper substrate
 Modification of submerged morphology
 Elimination of unwanted compounds
 Catabolite derepression
 Phosphate deregulation
 Permeability alterations to improve product export rate
 Metabolic resistance
 Production of
 Additional enzymes
 Compounds to inhibit contaminant microorganisms
 heterologous proteins that may also be engineered with downstream
processing ‘aids’, e.g. polyarginine tails

12. INDUSTRIAL STRAIN
IMPROVEMENT
 Approaches of strain improvement
 Creation of recombinant/mutants
 Three methods
 Natural recombination
 Mutagenesis
 Recombinant DNA Technology/Genetic Engineering
 Screening
 Storing in specific media for stability

13. INDUSTRIAL STRAIN
IMPROVEMENT
 Natural recombination
 New gene by combining from different straits
 Bacterial DNA= single chromosome + Plasmids
 Autonomous self-replicating accessory piece of DNA
 Plasmid carries up to a few 100 additional genes
 1000 copies of a plasmid/cell
 Contains supplemental genetic information coding
 Bacteria have no sexual reproduction
 Exchange of genetic information through plasmids

14. INDUSTRIAL STRAIN
IMPROVEMENT
 Process of natural recombination
 Cross-over
 Exchange of genetic materials between two chromosomes
 Conjugation
 Transduction
 Transformation
 Protoplast fusion

15. INDUSTRIAL STRAIN
IMPROVEMENT
 Process of natural recombination
 Cross-over
 Conjugation
Cell (D) to cell (R) contacts
With filamentous protein
called a sex pilus
It draws two cells together
 Transduction
 Transformation
 Protoplast fusion

16. INDUSTRIAL STRAIN
IMPROVEMENT
 Process of natural
recombination
 Cross-over
 Conjugation
 Transduction
 Transfer gene between two
cells through bacteriophase
 It attaches to a bacterial cell
 Injects its DNA into the
host
 Transformation
 Protoplast fusion

17. INDUSTRIAL STRAIN
IMPROVEMENT
 Process of natural
recombination
 Cross-over
 Conjugation
 Transduction
 Transformation
 cellular uptake of a naked
piece of DNA from the
surrounding medium
 It is random in nature
 Competent cell are only
possible to enter
 Protoplast fusion

18. INDUSTRIAL STRAIN
IMPROVEMENT
 Process of natural
recombination
 Cross-over
 Conjugation
 Transduction
 Transformation
 Protoplast fusion
 The fusion between non-
producing strains and yielded a
new strain.
 Losing dividing cell membrane

19. INDUSTRIAL STRAIN
IMPROVEMENT
 Mutagenesis
 Process of changing or creating genetic
information into the DNA of a cell
 Changes may be deletion, insertion,
duplication, inversion and
translocation of a piece of DNA or
 a change in the number of copies of an
entire gene or chromosome
 a very effective tool in improving
many industrial microorganisms.
 Mutants are considered to be the
product of natural events
 There are fewer problems in gaining
approval from regulatory body

20. INDUSTRIAL STRAIN
IMPROVEMENT
 Types of Mutagenesis
 Mutation are:
 Spontaneous mutation
 Induced mutation
 Directed mutation/ Site
directed mutation

21. INDUSTRIAL STRAIN
IMPROVEMENT
 Spontaneous Mutation
 Occurs naturally due to
unknown reason
 Rate is very low
 10-10
to 10-15
per generation per
gene
 Also called cellular
abnormalities
 Error in replication like
mismatch, insertion or deletion
etc
 Occurs random

22. INDUSTRIAL STRAIN
IMPROVEMENT
 Induce mutation
 Mutation occurs due to action of any
agents or factors
 Called Mutagens
 Rate greatly increased
 Mutagens are two types:
 Physical
 ultraviolet, ϒ and X radiation
 Chemical
 Ethane methane sulphonate (EMS), nitroso methyl
guanidine (NTG), nitrous acid and acridine
mustards
 Mutants occurs when changes in base sequence
of DNA
 Like basepair substitutions, frame-shift
mutations or large deletions that go unrepaired
 This is not specific rather random
 Improvement occurs randomly either lose of
any undesirable character

23. INDUSTRIAL STRAIN
IMPROVEMENT
 Directed mutation
 Site-directed mutagenesis
 Intentional changes to the DNA
sequence of a gene
 Basic mechanism
 Synthesis of a short DNA primer
 Primer contains desire mutation
 It must be complementary to template
DNA around the mutation site
 Primer is then hybridized with the DNA
in the gene of interest
 Primer is then extended using DNA
polymerase
 Copies the rest of the gene
 The gene thus copied contains the
mutated site,
 Then introduced into a host cell as a
vector and cloned
 Finally mutants are selected using DNA
sequencing

24. INDUSTRIAL STRAIN
IMPROVEMENT
 Approaches of SDM
 Kunkel's method
 Cassette mutagenesis
 PCR site-directed mutagenesis
 Whole plasmid mutagenesis
 In vivo site-directed mutagenesis methods
Before proceeding, let us
introduce with gene and
genetic map

25. INDUSTRIAL STRAIN
IMPROVEMENT
Prokaryotic
Eukaryotic
 Hereditary information is stored in and transformed
from cell
 Cells contain Nucleus having chromosome
 Composition of chromosome is about 40% DNA and
60% protein.
 DNA is found as the storehouse of all Hereditary
information.

26. INDUSTRIAL STRAIN
IMPROVEMENT
 DNA
 Composed of subunits called nucleotides.
 Each nucleotide is composed of three subunits:
 A pentose sugar; deoxyribose
 One of four nitrogenous bases; and
 A phosphate group
 These subunits always bond in the same way to make a complete nucleotide
 Each nucleotide is identified by the particular base which it contains.
 Four bases found in nucleotides are
 Adenine
 Thymine
 Guanine
 Cytosine
 Four nucleotides make up the vast majority of DNA molecules

27. INDUSTRIAL STRAIN
IMPROVEMENT
 DNA
 Nucleotides are arranged in a
long, straight strands called poly-
nuleotide strand
 Formed by covalent bond between
sugar and phosphate group
 Phosphate group of one
nucleotide is attached to the no 3
carbon (3′-C) of the preceding
nucleotide’s deoxyribose and the
no 5 carbon (5′-C) of the
succeeding nucleotide’s
deoxyribose.

28. INDUSTRIAL STRAIN
IMPROVEMENT
 DNA
 DNA molecule is made up of two
polynucleotide strands
 Both are twisted around one another
 This spiral structure is called a double helix
 Two strands runs in opposite directions
anti-parallel.
 Interior contains base pairing joining by H-
bonds
 Pairing occurs between one purine and one
pyrimidine base
 Thymine with adenine (2-H bonds)
 Guanine with cytosine (3-H bonds)
 Four base pairing are possible
 Base in one strand deduce the sequence of
others
 Complementary

29. INDUSTRIAL STRAIN
IMPROVEMENT
 The processes of development, growth, and repair of
an organism involve cell division.
 Chromosomal materials inside the cells require to go
double before actual splitting.
 Complementary structure DNA make it to occur
 Base sequence of on one strand allows to predict the
base sequence of the other strand
 One strand acts as template
 New molecules of DNA produced by DNA replication
 DNA replication occurs by three stages

30. INDUSTRIAL STRAIN
IMPROVEMENT
 Class practice
 Write the four possible base pairing.
 Write the complementary pairing of AATCGTCG

31. INDUSTRIAL STRAIN
IMPROVEMENT
 Living beings are made up with different polypeptides.
 The DNA contains all information necessary to
construct all polypeptides.
 Each individual information is called gene.
 Gene copies in the form of a molecule called
messenger RNA or mRNA.
 The workplace of mRNA is ribosome in cytoplasm.
 During the work tRNA works as labour.
 Both DNA and RNA have structural similarities and
dissimilarities.
 mRNA contained hereditary information copied from
DNA.
 Polypeptide Gene  mRNA  DNA

32.  The process of copying information from DNA to
RNA is called Transcription
 Steps of transcription
 Unwinding
 Complementary base pairing
 Elongation: RNA polymerase
 separation
INDUSTRIAL STRAIN
IMPROVEMENT

33.  Language of RNA
 Alphabets of RNA language are bases (A, U, G, C).
 Words are made with three bases.
 Each word is called a Codon.
 There are 64 Codons.
 Since only sixty four combination is possible of four
letters into three letters words.
 There are star and stop codon
 No codon specifies more than one AA
INDUSTRIAL STRAIN
IMPROVEMENT

34. COMPLETE PROTEIN
SYNTHESIS
 Information contained in the RNA is translated
to define its structure and function.
 Three steps
 Initiation
 Elongation
 Termination

35. INDUSTRIAL STRAIN
IMPROVEMENT
 Approaches of SDM
 Kunkel's method
 Cassette mutagenesis
 PCR site-directed mutagenesis
 Whole plasmid mutagenesis
 In vivo site-directed mutagenesis methods

36. MICROBIAL METABOLISM
 Energy yielding compounds
 Metabolism encompasses all enzyme catalyzed
reactions of a cell
 All enzyme catalyzed reactions are classified as
 Primary processes
 Secondary processes
 Primary metabolic pathways are largely common to most
organisms
 They involve both energy-generating metabolism called
 Catabolism
 Anabolism
 They utilize energy in the biosynthesis of cellular
components for growth and maintenance

37. MICROBIAL METABOLISM
 Energy yielding compounds
 Products of primary metabolisms
 Industrially important
 Use growth and development
 alcohols, amino acids, organic acids, nucleotides, enzymes and
microbial cells (biomass).
 Products of Secondary metabolisms
 Produces diverse products
 often species-specific end-products
 They are not used during rapid growth
 Many industrially important secondary metabolites include
alkaloids, antibiotics, toxins and some pigments

38. MICROBIAL METABOLISM
 Energy yielding compounds
 Catabolism
 All vegetative microbial cells require a continuous
supply of energy
 MOs get energy from organic sources
 Then transformed by an ordered series of enzyme-
controlled reactions within specific metabolic
pathways.
 This breakdown metabolism (catabolism) leads to the
generation of potential energy
 In the form of adenosine 5′-triphosphate (ATP) and
reduced coenzymes and heat
 Reduced enzymes are NADH, NADPH, FADH2