David Shonnard Department of Chemical Engineering Michigan ...
[Pages:25]Chapter 6: How Cells Grow
David Shonnard Department of Chemical Engineering
Michigan Technological University
1
David R. Shonnard
Michigan Technological University
Presentation Outline:
G Introduction G Batch Growth Characteristics
Growth Stages, Effects of Environmental Conditions, Product Formation, Mathematical Models
G Continuous Growth Characteristics
Dilution Rate, Optimum Operation
2
David R. Shonnard
Michigan Technological University
1
Introduction
Cell growth is the primary response of viable cells to substrates and nutrients.
Substrates/nutrients + cells products + more cells
specific growth rate (h-1), ? 1 dX X dt
X = cell mass concentration (g / L) t = time (h)
Product formation is a secondary response.
3
David R. Shonnard
Michigan Technological University
Determining Cell Concentration
1. Cell number concentration
a) hemocytometer (Petroff-Hausser slide) b) viable cell counts (petri dish) c) electronic particle counter
"Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
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David R. Shonnard
Michigan Technological University
2
Determining Cell Concentration (cont.)
2. Cell mass concentration
a) direct methods dry weight (filtration or centrifugation) packed cell volume (centrifugation) optical density (light scattering, 600-700 nm)
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David R. Shonnard
Michigan Technological University
Determining Cell Concentration (cont.)
2. Cell mass concentration (cont.)
a) indirect methods
measure biomolecule concentration and correlate to dry cell mass concentration.
(DNA, protein, ATP, NADH, product formation)
Example 1. NH4+ utilization during growth releases H+, amount of OH- added is proportional to growth.
Example 2. Luciferin + O2 + ATP
light
luciferase
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David R. Shonnard
Michigan Technological University
3
Batch Growth Curve
Inoculum [Xo]
[So]
growth medium (substrate + nutrients)
Batch Reactor
X = cell Concentration (cells/mL)
"Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
7
David R. Shonnard
Michigan Technological University
Lag Phase
no increase in cell numbers induction of enzymes to utilized substrate(s) very important to decrease lag period to productivity
i. Inoculate with exponential phase cells ii. Pre-acclimate inoculum in growth media iii. Use high cell inoculum size (5-10% by volume)
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David R. Shonnard
Michigan Technological University
4
Exponential Growth Phase
1. Nutrient and substrate concentrations are large
2. Growth rate is independent of nutrient and substrate conc.
3. Cell number and mass concentrations increase exponentially
dX dt
=
?max X, X = Xo at t =
0
X
=
X e? max o
t
or
X ln
Xo
= ?max t
doubling time of cells (td ),
X Xo
=2
ln 2 td = ?max
or
? max =
ln 2 td
X ln
Xo
o
o
o
o
Slope = ?max
t
ln(2) = ?max td
4. Balanced growth occurs cell composition constant
9
David R. Shonnard
Michigan Technological University
Deceleration Phase
depletion of one or more nutrients accumulation of toxic byproducts of growth unbalanced growth and metabolism shifts for survival
10
David R. Shonnard
Michigan Technological University
5
Stationary Phase
no net growth of cell numbers or cell mass (no cell division) cell growth rate = cell death rate secondary metabolites (products) produced endogenous metabolism of energy stores can result in maintaining cell viability removal of inhibitory compounds will result in further growth if additional substrate is provided
11
David R. Shonnard
Michigan Technological University
Death Phase
1. Cell lysis (spillage) may occur 2. Rate of cell decline is first-order
dX dt
=
- kd' t, X = XS at t =
0
X = X e-kd' t or ln X = - k ' t
S
Xo
d
3. Growth can be re-established by transferring to fresh media
12
David R. Shonnard
Michigan Technological University
6
Effects of Temperature on Cell Growth
?max doubles for each 10 T ?8?vprhr?rh? opt
(?max - kd' )
?max = A e-Ea / RT and kd' = A e' -Ed / RT Ea = activation energy for growth
10 to 20 kcal / mole Ed = activation energy for death
60 to 80 kcal / mole
As T , kd' faster than ?max, (?max- kd')
"Bioprocess Engineering: Basic Concepts, Shuler and Kargi, Prentice Hall, 2002
13
David R. Shonnard
Michigan Technological University
pH Effects
acceptable pH is ? 1 to 2 pH units
pH range varies by organism
bacteria (most) pH = 3 to 8
yeast
pH = 3 to 6
plants
pH = 5 to 6
animals
pH = 6.5 to 7.5
microorganism have the ability to control pH inside the cell, but this requires maintainance energy
pH can change due to
? utilization of substrates; NH4+ releases H+, NO3- consumes H+ ? production of organic acids, amino acids, CO2, bases
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David R. Shonnard
Michigan Technological University
7
pH Effects (cont.)
"Bioprocess Engineering: Basic Concepts
Shuler and Kargi, Prentice Hall, 2002
15
David R. Shonnard
Michigan Technological University
Dissolved O2 Effects
O2 may be a limiting substrate for aerobic fermentation, since O2 is sparingly soluble in water
critical O2 concentration 5 to 10% of saturation (? 7 mg O2/L) for bacteria/yeast
growth exhibits saturation kinetics with respect to O2 concentration (see next page)
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David R. Shonnard
Michigan Technological University
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