Batch culture: Growth Kinetics

m = m max s (Ks +s)

m max

1/2 m max Ks = substrate concentration

m= specific growth rate

Residual substrate conc. [s]

m

During log phase growth reaches maximum (max)After depletion of substrate, growth rate decreases and finally ceases

As growth increases biomass increases: during log phase

dx = mxdt

dx. 1 = mdt x

x = cell conc (biomass) (mg/m3)t = incubation time (h)m = specific growth rate (h-1)

x

dxdt m =slope

1

Beginning of log phase t=0 biomass X0

On integration of equation 1

∫dx = ∫ mx x

Loge X = mt + K (integration constant)

when t=0

Log X0 = K put this value in equation 2

loge X = mt + loge X0

Loge X –loge X0 = mt

ln X = mt X0

ln X . 1 = td X0 m

2

3

When t = tdX = 2X0

Then ln X . 1= td X0 m

ln 2X0 . 1 = td X0 m

ln 2 = td m

0.693 = td m m = 0.693

td

m is inversely proportional to td

If td is high m is low and vice versa

X0 cells inoculated at time t0X cells at time t

dx = mx Can be written as equation 3dt ln X = mt

X0

ln X –ln X0 = mt

Converting natural log

(log10 X –log10 X0) 2.303 = mt

(log10 X –log10 X0) 2.303 = tt-t0 m

(log10 X –log10 X0) 2.303 = m tt-t0

m = m max s (Ks +s)

m max

1/2 m max Ks = substrate concentration

m= specific growth rate

Residual substrate conc. [s]

m

Continuous cultureVolume added should be volume removedV working volume of the fermenter: m3F rate of flow in and out m3h-1

Dilution rate = F/V

F = DV (h-1)

Output of biomass in continuous cultureRate at which medium passes out of the outflow (flow rate F)conc of biomass in the outflow (i.e. X)

Output = FXSince F= DV Output = DVX

Productivity that is output per unit volumeprod = DVX prod = DX

V

Basic principles of continuous culture is controlled by Dilution rate

Rate of limiting substrate conc not m

Continuous enrichment culture

Continuous enrichment culture

MO isolated by this method survive fermentation much better than batch isolated MO

Main problem:Washout of the inoculum

Solution:

Isolate MO in a batch culture using 20% inoculum, as soon as growth is observed transfer to fresh medium so that stabilization and subsequent purification is performed in a continuous culture

Periodic inoculation of soil or sewage to the culture will ensure as the source of potential isolates; dominants must be resistant to contamination.

Measurement of Microbial Growth

Wet weight measurement

Dry weight measurement: 10-20% of wet weight

Absorbance: spectrophotometer

Total cell count: haemocytometer

Viable cell count: dilution plate method

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 productityIf 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 MOOccurs when all nutrients are provided in the mediumIts is essential for survival and existence of the organism and reproductionCells 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 is 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

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

METABOLIC PATHWAYS IN MICRO-ORGANISMS

1. PROVIDES PRECURSORS FOR THE CELL COMPONENTS2. ENERGY FOR ENERGY REQUIRING PROCESSES

Unique feature of heterotrophic MOSecrete extracellular enzymes

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 Pyruvate

(1) Glycolysis: glucose to pyruvate

6-carbon phase

oxidation phase

energy harvest phase

Glucose

Pyruvic acid

Glucose 6 Phosphate Pentose phosphate pathwayKDPGpathway

Glucose

Glucose-6-P

6-phosphogluconate instead of Fructose 6-P

2-keto-3-deoxy-6-phosphogluconate (KDPG)

Pyruvate glyceraldehyde-3-P

Embden-Meyerhof pathway

ATP

2ATP

NADPH

Entner-Doudoroff pathway

Pyruvate