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