2. Microbial Growth Kinetics

Report
Batch culture: Growth Kinetics
During log phase growth reaches maximum (max)
After depletion of substrate, growth rate decreases and finally ceases
m = m max s
(Ks +s)
m
m= specific growth rate
m max
1/2
m max
Ks = substrate concentration
Residual substrate conc. [s]
As growth increases biomass increases: during log phase
dx = mx
dt
1
dx. 1
dt x
x = cell conc (biomass) (mg/m3)
t = incubation time (h)
m = specific growth rate (h-1)
=m
dx
dt
m =slope
x
Beginning of log phase t=0 biomass X0
On integration of equation 1
∫dx
x
= ∫ mx
Loge X = mt + K (integration constant)
2
when t=0
Log X0 = K
put this value in equation 2
loge X = mt + loge X0
Loge X –loge X0 = mt
ln X
X0
= mt
ln X . 1 = td
X0 m
3
When
t = td
X = 2X0
Then
ln X . 1 = td
X0 m
ln 2X0 . 1
X0 m
ln 2
m
= td
= td
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 t0
X cells at time t
dx = mx
dt
Can be written as equation 3
ln X
X0
= mt
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= specific growth rate
m
m max
1/2
m max
Ks = substrate concentration
Residual substrate conc. [s]
Continuous culture
Continuous enrichment culture
Volume added should be volume removed
V working volume of the fermenter: m3
F rate of flow in and out m3h-1
Dilution rate = F/V
F = DV (h-1)
Basic principles of continuous culture is
controlled by Dilution rate
Rate of limiting substrate conc not m
Output of biomass in continuous culture
Rate at which medium passes out of the outflow (flow rate F)
conc of biomass in the outflow (i.e. X)
Output = FX
Since F= DV
Output = DVX
Productivity that is output per unit volume
prod = DVX
prod = DX
V
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
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 productity
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:
•
•
•
•
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
Unbranched pathway
intermediate
A ---- > B ----> C -----> D ------> E
Starting
substrate
Final end prod
Blocked reaction
Required metabolite
SECONDARY METABOLITES
• 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
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
METABOLIC PATHWAYS IN MICRO-ORGANISMS
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
6-carbon phase
oxidation
phase
energy harvest
phase
Glucose
KDPG
pathway
Glucose 6 Phosphate
Pyruvic acid
Pentose phosphate pathway
Entner-Doudoroff pathway
Glucose
ATP
Glucose-6-P
NADPH
6-phosphogluconate instead of Fructose 6-P
2-keto-3-deoxy-6-phosphogluconate (KDPG)
Pyruvate
glyceraldehyde-3-P
Meyerhof pathway
2ATP
Pyruvate
Embden-

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