Development of industrial fermentation processes
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Money making
Competition
Economically feasible on large scale basis
Recovery of product ready for open market
Competitive advantage
Criteria for being important in choice of organism
1. Nutritional characteristics of the organism when grown
on a cheap medium
2. Optimum temp of the organism
3. Reaction of the organism with the equipment and
suitability for the type of process
4. Stability of the organism and its amenability for genetic
manipulation
5. Productivity of the organism i.e. ability to convert
substrate into product per unit time
6. Ease of product recovery from the culture
What are the R&D approaches for finding of a MO of
economic value, and large scale fermentation
process?
Micro-organism
Stock culture
collections
Source
Screening
Primary screening
Secondary screening
Environment (soil)
Primary screening
• Highly selective procedures for detection and isolation of MO of
interest
• Few steps will allow elimination of valueless MO
• Eg. Crowded plate technique for Ab screening, serial dilution, acid base
indicator dyes, CaCO3, sole source carbon or nitrogen, enrichment tech
• Does not give too much information on detail ability of the microorganisms
• May yield only a few organisms and few of them may have commercial
value
Common techniques
I. 1.
2.
3.
4.
5.
6.
Direct wipe or sponge of the soil
Soil dilution (10-1 to 10-10)
Gradient plate method (streak, pour)
Aerosol dilution
Flotation
Centrifugation
II. Enrichment, screening for metabolites or microbial products
III. Unusual environments
Secondary screening
• Sorting of MO that have real commercial value for industrial
processes and discarding those which lack potential
• Conducted on agar plates (not sensitive), small flasks or small
fermentors (more sensitive) containing liquid media or
combination of these approaches.
• Liquid culture provide better info on nutritional, physical and
production responses.
• Can be qualitative or quantitative
Preservation of Industrially important MO
• Viable and Free from contamination
• Stored in such a way so as to eliminate genetic change and retain
viability
• Viable by repeated sub-culture (avoid mutations by keeping stocks
and strain degeneration and contaminations)
Preservation of Industrially important MO
1. Storage at reduced temperature
a.
Agar slopes at 50C or in -200C freezer: viable for 6 months
b. Liquid nitrogen (-1960C): problems of refilling, advantages
2. Storage at dehydrated form
a.
Dried cultures
b. Lyophillization
Quality control of preserved stock: batch system, single colony,
typical pattern, large number, purity, viability and productivity
If sample fails entire batch is destroyed
MICROBIAL METABOLIC PRODUCTS OR METABOLITES
• Wide range of products having commercial value
Algae
SCP
Bacteria
acetic acid
bactracin
gramicidin
endotoxin
glutamic acid
vitamin B12
Actinomycetes
antibiotics (tetracycline,
streptomycin, neomycin, rifamycin,
gentamycin)
Fungi
citric acid, amylase, cellulase, SCP,
lipase, pencillin, ethanol, wine,
steroids, gibberllin
TYPES OF LOW MOLECULAR WEIGHT COMPOUNDS BY MO
SUBSTRATE
Primary
metabolites
Secondary
metabolites
Antibiotics
Essential metabolites
Amino acids
Nucleosides
vitamins
Ethanol, acetone, lactic
acid, butanol
Steroids
Amino acids
Alkaloids
Gibberlins
Pigments
Metabolic end products
Bioconversions
Ascorbic acid
Primary metabolism
Secondary metabolism
Idiophase
Trophophase
Concentration
Cell Mass
Limiting
nutrient
Secondary
metabolite
Time
PRIMARY METABOLITES
 Formed in trophophase (log phase)
 Balanced growth of MO
 Occurs when all nutrients are provided in the medium
 Its is essential for survival and existence of the organism and
reproduction
 Cells have optimum concentration of all macromolecules (proteins,
DNA, RNA etc.)
 Exponential growth
PRIMARY METABOLITES
1.
Primary essential metabolites:
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Produced in adequate amount to sustain cell growth
Vitamins, amino acids, nucleosides
These are not overproduced, wasteful
Overproduction can be genetically manipulated
2. Primary essential end products:
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Normal end products of fermentation process of primary
metabolism
Not have a significant function in MO but have industrial
applications
Ethanol, acetone, lactic acid, CO2
LIMITATIONS:
growth rate slows down due to limited supply of any
other nutrient. Metabolism does not stop but
product formation stops.
OVERPRODUCTION OF PRIMARY METABOLITES
Manipulation of feedback inhibition
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Auxotrophic mutants having a block in steps of a biosynthetic pathway
for the formation of primary metabolite (intermediate not final end
prod).
End product formation is blocked and no feedback inhibition
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Mutant MO with defective metabolite production
Unbranched pathway
intermediate
A ---- > B ----> C -----> D ------> E
Starting
substrate
Final end prod
Blocked reaction
Required metabolite
SECONDARY METABOLITES
idiophase
• Characterized by secondary metabolism and secondary metabolites (idolites)
• Produced in abundance, industrially important
Characteristics:
1.
2.
3.
4.
Specifically produced
Non essential for growth
Influenced by environmental factors
Some produce a group of compds eg a strain of Streptomyces
produced 35 anthracyclines
5. Biosynthetic pathways are not established
6. Regulation of formation is more complex
Functions:
1. May or may not contribute for existence or survival of the MO
OVERPRODUCTION OF SECONDARY METABOLITES
More complex
Several genes are involved eg may be 300 to 2000 genes
Regulatory systems are more complex
Some regulatory mechanisms
1. Induction: eg tryptophan for ergot production etc
2. End product regulation: some metabolite inhibit their own biosysnthesis
3. Catabolite regulation: key enzyme inactivated, inhibited or repressed
eg. Glucose can inhibit several antibiotics
ammonia as inhibitor for antibiotic prod.
4. Phosphate regulation: Pi for growth and multiplication in pro and
eukaryotes. Increase in pi conc can increase secondary metabolites but
excess harmful
5. Autoregulation: self regulation mechanism for production like hormones
BIOCONVERSIONS OR BIOTRANSFORMATIONS
Used for chemical transformation of unusual substrates for desired prods
Conversion of
ethanol to acetic acid,
sorbitol to sorbose,
synthesis of steroid hormones and
certain amino acids
Structurally related compounds in one or few enzymatic reactions
Can use resting cells, spores or even killed cells.
Mixed cultures can also be used, use of immobilized cells at low cost
?
BIOCONVERSIONS OR BIOTRANSFORMATIONS (BTs)
When and why is biotransformation done?
 when production of a particular compound is difficult or costly by chemical
methods
 BTs are preferred over chemical reactions due to substrate specificity,
stereospecificity, mixed reaction conditions (pH, temp, pressure)
 Environmental pollution is negligible
 Easy to apply recombinant DNA technology
 Easy to scale up the processes sue to limited number of reactions
TYPES OF HIGH MOLECULAR WEIGHT COMPOUNDS BY MO
 Polysaccharides, proteins (enzymes)
 Pharmaceutical products
Enzymes
 naturally occurring biocatalysts; accelerate metabolic reactions
 Production of primary and secondary metabolites are not possible without
enzymes
 Enzymes during fermentation are EXTRACELLULAR (amylase, cellulase, lipase,
b-galactosidase, esterase, protease, chitinase, xylanase, glucose isomerase)
and some are INTRACELLULAR (invertase, asparginase)
 Extremozymes
 Immobilized enzymes
Microbial Biomass
 Microbes can themselves be products or main source of biomass
 Microbial biomass is exploited as microbial protein or single cell
protein (SCP)
METABOLIC PATHWAYS IN MICRO-ORGANISMS
1. PROVIDES PRECURSORS FOR THE CELL COMPONENTS
2. ENERGY FOR ENERGY REQUIRING PROCESSES
Unique feature of heterotrophic MO
Secrete extracellular enzymes
1. Catabolism
2. Amphibolism (Intermediate metabolism requiring central
metabolic pathways)
3. Anabolism
4. Function of enzymes: substrate specificity, catalysis
5. Coenzymes and prosthetic group
6. Methods of ATP generation: SLP, OP (respy), OP (photosyn)
7. Uptake of substrates (diffusion, FD, AT, Gp Trans,
siderophores
8. Degradation of carbon and energy sources (sugar breakdown)
METABOLIC PATHWAYS IN MICRO-ORGANISMS
Carbon and energy source breakdown
Sugars to Pyruvate
The ways in which microorganisms degrade sugars to
pyruvate and similar intermediates are introduced by
focusing on only three routes:
(1) Glycolysis (Embden Meyerhof Pathway)
(2) The pentose phosphate pathway,
(3) The Entner-Doudoroff pathway
(1) Glycolysis: glucose to pyruvate
Hexokinase
6-carbon phase
phosphofructokinase
Fructose biphosphate aldolase
oxidation
phase
energy harvest
phase
Glucose
KDPG
Pathway
Or
Entner
Dourdoff
Pathway
Glucose 6 Phosphate
Pyruvic acid
Acetyl CoA
Pentose phosphate pathway
Centre of
Intermediate
metabolism
Precursor for
Numerous
Biosynthetic pathways
(2) Entner-Doudoroff pathway or KDPG pathway
Glucose ----> 2 pyruvic acid + ATP +NAD(P)H2 + NADH2
Glucose
6 phosphogluconate dehydrase
Glucose-6-P
ATP
6 Phosphogluconolactone
NADPH
6-phosphogluconate
instead of Fructose 6-P
2-keto-3-deoxyphosphogluconate aldolase
2-keto-3-deoxy-6-phosphogluconate (KDPG)
Embden-Meyerhof pathway
Pyruvate
glyceraldehyde-3-P
Pyruvate
Only in prokaryotes, many gram negative bacteria some G+ve
Operates when glycolytic enzymes like phosphofructokinase-1 are
lacking
1 net ATP is produced
1 NADPH and 1 NADH is also produced
2ATP
1 NADH
(3) Pentose Phosphate pathway (PPP) or HMP
 Heterofermenter lactobacilli
 Bacteria which lack aldolase for conversion to triose phosphate
 PPP takes place
Oxidative catabolism of glucose
Dehydrogenation
hydrolysis
Reducing equivalents
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Glucose 6 phosphate
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*
glycolysis
To
Microbial fermentation pathways
CO2
LAF
NADH
acetaldehyde
EthanolAF
MxAF
BuDF
BuAF
PropAF
BuAcetoneF
BuAcidF
METABOLIC PATHWAYS IN MICRO-ORGANISMS
Precursors for
biosynthesis
3C
2C
4C
NADH
6C
4C
6C
4C
5C
4C
GTP
CO2
4C
CO2
NADH
CO2
NADH
LMW constituents
Glycolysis
Pentose phosphate pathway
Entner Douordoff pathway
Krebs cycle
Purines
Pyrimidines
Lipids
Phospholipids
Amino acids
Macromolecular constituents
Glycolysis
Pentose phosphate pathway
Entner Douordoff pathway
Krebs cycle
DNA, RNA
Proteins
Peptidoglycans
Polysaccharides (glycogen, starch, PHB)
Glucose
ATP
PPP
G-6-P
AA syn
Histidine
Tryptophan
Ribose 5P
Tyrosine
Erythrose 4P
Phenylalanine
pyruvate
Gly
Serine
Cys
Ala
Leu
Oxaloacetate
Val
Lys
a keto glutarate
Glu --- Gln
Pro
AROMATIC
AA
GLUTAMATE
FAMILY
Ori -- citruline -- Arg
DAP
Asp
SERINE
FAMILY
PYRUVATE
FAMILY
ASPARTATE
FAMILY
Asn
Homoserine - Met
Thr ----- Ile