Microbial Growth

ChE 3BK3
October 1, 2004
Chapter 6, Shuler & Kargi
Outline
z Effect of environmental conditions on
growth kinetics
pH
Temperature
Dissolved oxygen
z Heat generation by microbial growth
Environmental Factors
z Patterns of microbial growth and product
formation are influenced by environmental
factors such as temperature, pH and dissolved
oxygen concentration (D.O.)
z Microorganisms can be classified by their
optimum growth temperatures, Topt
Psychrophiles: (Topt < 20oC)
Mesophiles: (20oC < Topt < 50oC)
Thermophiles: (Topt > 50oC)
z As the temperature increases towards Topt, the
growth rate doubles every ~10oC
Optimum Growth Temperature
Optimum Growth Temperature
Temperature
z Above Topt the growth rate decreases and
thermal death may occur
The net specific replication rate for temperatures above
Topt is expressed by:
dN
dt
( )
= R' k d' N (6.19)

z Both R' and k d' vary with temperature

according to the Arrhenius equation:
' Ea / RT
= Ae
'
R
E a / RT
and k = Ae
'
d
(6.20)
Arrhenius Plot of Growth Rate of
E. Coli B/r

Legend:
() Growth on rich,
complex medium
() Growth on glucose-
mineral salts medium
pH
z pH affects the activity of enzymes, and
therefore the microbial growth rate
z The optimal pH for growth may be different
from the optimal pH for product formation (e.g.
Pichia pastoris)
z Acceptable pHs for growth are typically within
1 or 2 pH units of the optimum pH
z Bacteria (3 - 8), yeast (3 - 6) and fungi (3 - 7)
tend to have wider acceptable pH ranges than
plant (5 - 6) and animal cells (6.5 - 7.5)
Typical Variation of Specific
Growth Rate with pH
Dissolved Oxygen
z At high cell concentrations, the rate of oxygen
consumption may exceed the rate of O2 supply
When oxygen is the rate-limiting factor, specific growth
rate varies with [DO] according to saturation
(Michaelis-Menten) kinetics
z Below a critical concentration, growth
approaches a first-order rate dependence on
DO (oxygen is a limiting substrate)
z Above a critical concentration, the growth rate
becomes independent of DO (oxygen is non-
limiting)
Growth Dependence on D.O.

Strict aerobe

Facultative aerobe
Dissolved Oxygen
z The saturated DO concentration for water at
25oC and 1 atm is ~7 ppm
The presence of dissolved salts and organics can alter
the saturation value
Increasing temperatures decrease the saturation value
z The critical oxygen concentration is about 5%-
10% of the saturated DO concentration for
bacteria and yeast, and about 10%-50% of
[DO]sat for moulds, since they grow as large
spheres in suspended culture (diffusion issues)
Dissolved Oxygen
z Oxygen is typically introduced into the fermentation
broth by sparging air through the broth
Oxygen transfer from gas bubbles to cells is usually limited
by O2 transfer through the liquid film surrounding the gas
bubbles
The rate of O2 transfer from the gas to the liquid phase is
given by:
( )
N O2 = k L a C C L = OTR
* (6.21)
Where kL is the oxygen transfer coefficient (cm/h), a is the
gas-liquid interfacial area (cm2/cm3), kLa is the volumetric
oxygen transfer coefficient (h-1), C* is the saturated DO
concentration (mg/l), CL is the actual DO concentration in the
broth (mg/l), and NO is the oxygen transfer rate (OTR: mg
2
O2/l-h)
Dissolved Oxygen
z The rate of oxygen uptake (OUR) is:
g X
OUR = qO2 X = (6.22)
YX / O2
z Where qO is the specific rate of oxygen
2
consumption (mg O2/g DCW-h), YX/O is the
2
yield coefficient on oxygen (g DCW/g O2), and
X is cell concentration (g DCW/l)
Dissolved Oxygen
z When oxygen transfer is the rate-limiting step,
the rate of oxygen consumption is equal to the
rate of oxygen transfer:

g X
= k L a (C C L )
*
(6.23)
YX O2
dX
or, = YX O2 k L a (C C L )
*
(6.24)
dt
Substrate Concentration
z Substrate concentrations significantly
above stoichiometric requirements are
inhibitory to cellular functions
Inhibitory levels of substrates vary depending
on cell type and substrate
Typical maximum non-inhibitory
concentrations of some nutrients are
glucose, 100 g/l; ethanol, 50 g/l for yeast,
much less for other organisms; ammonium,
5 g/l; phosphate, 10 g/l; nitrate, 5 g/l
Other Factors
z The redox potential is an important parameter
that affects the rate and extent of many
oxidative-reductive reactions
In fermentation media, the redox potential is a
complex function of DO, pH, and other ion
concentrations, such as reducing and oxidizing agents
z Dissolved CO2 can have a profound effect on
the performance of microorganisms
Very high DCO2 concentrations can be toxic to some
cells
On the other hand, cells require a certain minimum
DCO2 level for proper metabolic function
Heat Generation by Microbial
Growth
z About 40% to 50% of the energy stored in a carbon and
energy source is converted to biological energy (ATP)
during aerobic metabolism, and the rest of the energy is
released as heat
For actively growing cells, the maintenance requirement is
low, and heat evolution is directly related to growth
The heat of combustion of the substrate is equal to the sum of
the metabolic heat and the heat of combustion of the cellular
material:
H S 1
= H C + (6.27a)
YX S YH
Where HS is the heat of combustion of the substrate (kJ/g
substrate), HC is the heat of combustion of cells, and 1/YH is
the metabolic heat evolved per gram of cell mass produced
(kJ/g cells)
Heat Balance on Microbial
Utilization of Substrate
Heat Generation by Microbial
Growth
z Equation 6.27a can be rearranged to yield:
YX S
YH = (6.27b)
H S YX S H C
z HS and HC can be determined from the
combustion of substrate and cells
Typical HC values for bacterial cells are 20-25 kJ/g
cells
Typical values of YH are: glucose, 0.4 g/kcal; malate, 0.3
g/kcal; acetate, 0.21 g/kcal; ethanol, 0.18 g/kcal;
methanol, 0.12 g/kcal; and methane, 0.061 g/kcal
Clearly, the degree of oxidation of the substrate has a
strong effect on the amount of heat released
Heat Generation by Microbial
Growth
z The total rate of heat evolution in a batch
fermentation is:
1
QGR = VL net X (6.28)
YH
Where VL is the liquid volume (l)
z In aerobic fermentations, the rate of metabolic
heat evolution can roughly be correlated to the
rate of oxygen uptake:
QGR 0.12QO2 (6.29)

Where QGR is in kcal/h, and QO2 is in mM of O2/h

Heat Generation by Microbial
Growth
z Metabolic heat released during a
fermentation can be removed by
circulating cooling water through a
cooling coil within the fermenter, or a
cooling jacket surrounding the fermenter
z Temperature control is a critical limitation
on reactor design
z The ability to estimate heat removal is
essential to proper reactor design
Cooling Coils
Water-Jacketed Fermenter