development of industrial fermentation processes
DESCRIPTION
Development of industrial fermentation processes. 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. - PowerPoint PPT PresentationTRANSCRIPT
Development of industrial fermentation processes
• 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
Source Environment (soil)Stock culture collections
Screening
Primary screening
Secondary screening
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 micro-organisms
• May yield only a few organisms and few of them may have commercial value
Common techniques
1. Direct wipe or sponge of the soil2. Soil dilution (10-1 to 10-10)3. Gradient plate method (streak, pour)4. Aerosol dilution5. Flotation6. Centrifugation
I.
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 productivityIf sample fails entire batch is destroyed
MICROBIAL METABOLIC PRODUCTS OR METABOLITES
• Wide range of products having commercial value
Algae SCP
Bacteria acetic acidbactracingramicidinendotoxinglutamic
acidvitamin
B12Actinomycetes antibiotics (tetracycline, streptomycin, neomycin, rifamycin,
gentamycin)
Fungi citric acid, amylase, cellulase, SCP,
lipase, pencillin, ethanol, wine, steroids,
gibberllin
SUBSTRATE
Primary metabolites
Secondary metabolites
Bioconversions
Essential metabolitesAmino acidsNucleosidesvitamins
Metabolic end products
Ethanol, acetone, lactic acid, butanol
Antibiotics
Alkaloids
Gibberlins
Pigments
Steroids
Amino acids
Ascorbic acid
TYPES OF LOW MOLECULAR WEIGHT COMPOUNDS BY MO
Trophophase Idiophase
Limiting nutrient
Secondary metabolite
Cell Mass
Primary metabolism Secondary metabolism
Time
Concentration
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:
• 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:• 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• 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
• Mutant MO with defective metabolite production
A ---- > B ----> C -----> D ------> EFinal end prod
Required metabolite
Startingsubstrate
intermediate
Blocked reaction
Unbranched pathway
SECONDARY METABOLITES
• Characterized by secondary metabolism and secondary metabolites (idolites)
• Produced in abundance, industrially important
Characteristics:
1. Specifically produced2. Non essential for growth3. Influenced by environmental factors4. Some produce a group of compds eg a strain of Streptomyces
produced 35 anthracyclines5. Biosynthetic pathways are not established6. Regulation of formation is more complex
Functions:7. May or may not contribute for existence or survival of the MO
idiophase
OVERPRODUCTION OF SECONDARY METABOLITES
More complexSeveral genes are involved eg may be 300 to 2000 genesRegulatory 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 repressedeg. 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 MOSecrete extracellular enzymes
1. Catabolism2. Amphibolism (Intermediate metabolism requiring central
metabolic pathways)3. Anabolism4. Function of enzymes: substrate specificity, catalysis5. Coenzymes and prosthetic group6. Methods of ATP generation: SLP, OP (respy), OP
(photosyn)7. Uptake of substrates (diffusion, FD, AT, Gp Trans,
siderophores8. Degradation of carbon and energy sources (sugar
breakdown)
METABOLIC PATHWAYS IN MICRO-ORGANISMS
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
Sugars to PyruvateCarbon and energy source breakdown
(1) Glycolysis: glucose to pyruvate
6-carbon phase
oxidation phase
energy harvest phase
Hexokinase
phosphofructokinase
Fructose biphosphate aldolase
Glucose
Pyruvic acid
Glucose 6 Phosphate Pentose phosphate pathway
KDPGPathway
Or Entner
DourdoffPathway
Centre of Intermediate metabolism
Acetyl CoA Precursor for NumerousBiosynthetic pathways
Glucose
Glucose-6-P
6 Phosphogluconolactone
6-phosphogluconate instead of Fructose 6-P
2-keto-3-deoxy-6-phosphogluconate (KDPG)
Pyruvate glyceraldehyde-3-P
ATP
2ATP
NADPH
(2) Entner-Doudoroff pathway or KDPG pathway
PyruvateOnly in prokaryotes, many gram negative bacteria some G+veOperates when glycolytic enzymes like phosphofructokinase-1 are lacking1 net ATP is produced1 NADPH and 1 NADH is also produced
1 NADH
6 phosphogluconate dehydrase
2-keto-3-deoxyphosphogluconate aldolase
Glucose ----> 2 pyruvic acid + ATP +NAD(P)H2 + NADH2
Embden-Meyerhof pathway
(3) Pentose Phosphate pathway (PPP) or HMP
Heterofermenter lactobacilli
Bacteria which lack aldolase for conversion to triose phosphate
PPP takes place
Reducing equivalents
Dehydrogenationhydrolysis
Glucose 6 phosphate
Oxidative catabolism of glucose
*
To glycolysis
*
*
*
*
Microbial fermentation pathways
LAF
BuDFMxAF
acetaldehyde EthanolNADHCO2 AF
BuAcidF
BuAF
BuAcetoneFPropAF
METABOLIC PATHWAYS IN MICRO-ORGANISMS
2C
6C
6C
5C
4C
4C
4C
4C
4C
3C
CO2NADH
CO2NADH
CO2
Precursors for biosynthesis
NADH
GTP
Macromolecular constituents
DNA, RNAProteinsPeptidoglycansPolysaccharides (glycogen, starch, PHB)
LMW constituents
PurinesPyrimidinesLipidsPhospholipidsAmino acids
GlycolysisPentose phosphate pathwayEntner Douordoff pathwayKrebs cycle
GlycolysisPentose phosphate pathwayEntner Douordoff pathwayKrebs cycle
Glucose
G-6-P
pyruvate
Oxaloacetate
a keto glutarate
PPP
Ribose 5P
Erythrose 4P
Histidine
Tryptophan
Tyrosine
Phenylalanine
AROMATIC AA
Serine Gly
CysAlaLeu
Val
SERINE FAMILY
PYRUVATE FAMILY
ATP
Lys
DAP
Asp Asn Homoserine - Met
Thr ----- Ile
ASPARTATE FAMILY
Glu --- Gln Pro
Ori -- citruline -- Arg
GLUTAMATE FAMILY
AA syn