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