Microbial growth and growth

kinetics
 Objectives
1. The fastest known doubling time for a bacterium and under what
conditions this occurs
2. The slowest estimated doubling time for a bacterium and under
what conditions this occurs
3. Calculate a growth rate, u, from the slope of a growth curve
4. Compare and contrast growth in pure culture with growth in the
environment
5. The growth curve and the parts of the curve
6. A mathematical equation for each part of the curve as well as the
Monod equation
7. At least two electron acceptors that can be used under anaerobic
conditions in place of oxygen
8. Whether aerobic or anaerobic metabolism yields more energy
and why
9. The mass balance equation for aerobic metabolism
 Lecture 2 – Growth

What are the differences between growth in a flask in pure culture and
growth in the environment (e.g. soil, water, skin surfaces, leaf surfaces)?

vs.
 Growth Curve

9.0 Turbidity (optical density)

8.0
Stationary

Density
Log CFU/ml

Optical density
CFU/ml

7.0 De
a th
ia l
10

Optical
6.0
t
Log

onen
Ex p

5.0

4.0
Lag
Lag Time
Lag phase Three causes for lag: physiological lag
low initial numbers
appropriate gene(s) absent

growth approx. = 0 (dX/dt = 0)

Exponential phase
Nutrients and conditions are not limiting
growth = 2n or X = 2nX0
Where X0 = initial number of cells 20
X = final number of cells
21
n = number of generations

22

23

24

2n
Example: An experiment was performed in a lab flask growing cells on
0.1% salicylate and starting with 2.2 x 104 cells. As the experiment
below shows, at the end there were 3.8 x 109 cells.
This is an increase is 5 orders of magnitude!!

How many doublings or generations occurred?

1.0e+10

X = 2nX0
1.0e+9

Viable Count (CFU/ml)

3.8 x 109 = 2n(2.2 x 104) 1.0e+8

1.73 x 105 = 2n 1.0e+7

log(1.73 x 105) = nlog2 Cells grown on salicylate, 0.1%

1.0e+6
17.4 = n
1.0e+5

1.0e+4
0 20 40 60 80 100
Time (Hours)
 How does this compare to growth in the soil?

Response of culturable microbial community to addition of a

carbon source.
Unamended 1% Glucose
Soil CFU/g soil CFU/g soil Log Increase
Pima 5.6 x 105 4.6 x 107 1.9
Brazito 1.1 x 106 1.1 x 108 2.0
Clover Springs 1.4 x 107 1.9 x 108 1.1
Mt. Lemmon 1.4 x 106 8.3 x 107 1.7

Only a 1 to 2 order of increase!!

 Now compare how environmental conditions
can impact metabolism in soil

Degradation of straw under different conditions

Half-life u
Residue Days
Days-1 Relative rate

Wheat straw, laboratory 9 0.008 1

Rye straw, Nigeria 17 0.04 0.5
Rye straw, England 75 0.01 0.125
Wheat straw, Saskatoon 160 0.003 0.05
 Calculating growth rate during exponential growth

1.0e+10dX/dt = uX where u = specific growth rate (h-1)

Rearrange:
1.0e+9 dX/X = udt
Viable Count (CFU/ml)

Integrate:
1.0e+8 lnX = ut + C, where C = lnX0
dX/dt = uX
where u = specific growth rate (h-1)
1.0e+7
lnX = ut + ln X0 or X = X0eut

1.0e+6y = mx + b (equation for a straight line)

1.0e+5

Note that u, the growth rate, is the slope of this straight line
1.0e+4
0 20 40 60 80 100
Time (Hours)
 Calculating growth rate during exponential growth

dX/dt = uX where u = specific growth rate (h-1)

Rearrange: dX/X = udt

Integrate: lnX = ut + C, where C = lnX0

lnX = ut + ln X0 or X = X0eut

y = mx + b (equation for a straight line)

Note that u, the growth rate, is the slope of this straight line
 Find the slope of this growth curve

1.0e+10

1.0e+9
Viable Count (CFU/ml)

1.0e+8
lnX = ut + ln X0 or u = lnX – lnX0
1.0e+7 t – t0

1.0e+6 u = ln 5.5 x 108 – ln 1.7 x 105 = 2 hr-1

8.2 - 4.2
1.0e+5

1.0e+4
0 20 40 60 80 100
Time (Hours)
 Now calculate the doubling time

If you know the growth rate, u, you can calculate the doubling time
for the culture.
lnX = ut + ln X0

For X to be doubled: X/X0 = 2

or: 2 = eut

From the previous problem, u = 2 hr-1,

2 = e2(t)

t = 0.34 hr = 20.4 min

What is fastest known doubling time? Slowest?

 How can you change the growth rate???

When under ideal, nonlimiting conditions, the growth rate can only be
changed by changing the temperature (growth increases with increasing
temp.). Otherwise to change the growth rate, you must obtain a different
microbe or use a different substrate.

In the environment (non-ideal conditions), the growth rate can be

changed by figuring out what the limiting condition in that environment is.

Question: Is exponential growth a frequent occurrence in the

environment?
 Growth Curve

9.0 Turbidity (optical density)

8.0
Stationary
Stationary

Optical density
Log 10 CFU/ml

7.0 De
a th
ia l

6.0
t
onen
Ex p

5.0

4.0
Lag Time
 Stationary phase

nutrients become limiting and/or toxic waste products accumulate

growth = death (dX/dt = 0)

Death phase

death > growth (dX/dt = -kdX)

 Monod Equation
The exponential growth equation describes only a part of the growth curve
as shown in the graph below.

The Monod equation describes the dependence of the growth rate on the
substrate concentration:

u = um .S 1.0e+10

1.0e+9
Ks + S

Viable Count (CFU/ml)

1.0e+8

1.0e+7
u = specific growth rate (h-1)
1.0e+6

um = maximal growth rate (h-1) 1.0e+5

1.0e+4
S = substrate concentration (mg L-1) 0 20 40 60 80 100
Time (Hours)

Ks = half saturation constant (mg L-1)

Combining the Monod equation and the exponential growth equation allows
expression of an equation that describes the increase in cell mass through
the lag, exponential, and stationary phases of growth:

dX/dt = uX
u = um. S
Ks + S u = dX/Xdt
Monod equation Exponential growth equation

1.0e+10

1.0e+9

dX/dt = um. S . X Viable Count (CFU/ml) 1.0e+8

Ks + S 1.0e+7

1.0e+6

1.0e+5 Does not describe death phase!

1.0e+4
0 20 40 60 80 100
Time (Hours)
 There are two special cases for the Monod growth equation

1. At high substrate concentration when S>>Ks, the Monod equation

simplifies to:

dX/dt = umX

growth will occur at the

maximal growth rate.
Ks
2. At low substrate concentration
when S<< Ks, the Monod equation
simplifies to:

dX/dt = um. S . X
Ks
growth will have a first order dependence on substrate concentration
(growth rate is very sensitive to S).

Which of the above two cases is the norm for environmental samples?
 Growth in terms of substrate loss

In this case the growth equation must be expressed in terms of substrate

concentration. The equations for cell increase and substrate loss can be
related by the cell yield:
dS/dt = -1/Y (dX/dt) where Y = cell yield

Y = g cell mass produced

g substrate consumed

Glucose (C6H12O6) Pentachlorophenol (C6Cl5OH) Octadecane (C18H38)

0.4 0.05 1.49

 Growth in terms of substrate loss

dS/dt = -1/Y (dX/dt)

Combine with: dX/dt = um. S . X dS/dt = - um (S . X)

Ks + S Y (Ks + S)

Which parts of this curve does the equation describe?

phenanthrene (%)
Remaining

0 1 2 3 4 5 6 7 8
Time (days)
Aerobic vs. anaerobic metabolism
 Aerobic metabolism
General equation:
(C6H12O6) + 6(O2) 6(CO2) + 6(H2O)

Mass balance equation:

a(C6H12O6) + b(NH3) + c(O2) d(C5H7 NO2) + e(CO2) + f(H2O)
cell mass

The mass balance equation illustrates that some of the carbon in the
substrate is used to build new cell mass and some is oxidized completely
to CO2 to provide energy for the cell.

Using the mass balance equation and the cell yield, one can calculate the
% of the substrate carbon that is used to build new cell mass and the %
that is evolved as CO2

Examples of when this knowledge is important??

 Anaerobic metabolism
Under anaerobic conditions, the substrate undergoes
disproportionation, whereby some of the carbon is oxidized completely
to CO2 and some is reduced to CH4 (because CO2) acts as a terminal
electron acceptor.

General equation:
C6H12O6 + alternate TEA CO2 + CH4 + H2O

Some Typical Terminal Electron Acceptors

Form of Respiration Typical Redox Electron Products

Potential Acceptor

Aerobic respiration + 400 mV O2 H2 O

Nitrate respiration/denitrification - 100 mV NO3- NO2- , N2
Sulfate reduction - 160 to - 200 mV SO4-2 HS- , H2S
Methanogenesis - 300 mv CO2 CH4