Abstract
Wild type E. coli grows faster than both KO11 and LY01, reaching its peak optical
density after only 10 hours, and achieves a higher optical density than the other two.
Geneticly modified KO11 grows more slowly, taking 16 hours to finish growing, reaches
a lower optical density, and produces more ethanol than wild type E. coli. LY01 grows
more slowly than the other two, ceasing growing after 21 hours, and reaches the lowest
optical density of the three. It produces less ethanol than KO11 but more organic acids.
Introduction
As the fossil fuel reserves of the earth become depleted, more and more people are
turning to renewable fuel sources. One of these sources is plant biomass in the form of
ethanol. Escherichia coli produces ethanol from glucose in a fermentation process,
though not very efficiently. E. coli was genetically altered by adding genes for ethanol
production from Zymomonas mobilis. The resulting strain is known as KO11. This new
strain was subjected to increasing concentrations of ethanol. A mutant of KO11 able to
tolerate ethanol concentrations of up to 45 g/l was isolated. This bacterium is knows as
LY01.
The goal of this project is to study these three strains of E. coli. We will examine the
growth of these bacteria and the production of metabolites. The metabolic fluxes of
products (succinate, lactate, formate, acetate, and ethanol) can be combined with the
stoichiometry of the reactions involved to determine the movement of intermediates.
This knowledge can then be used to gain a better understanding of how the recombinant
bacteria function and can lead to better genetically altered strains.
Methods
A 700 ml stock solution of Luria-Bertani (LB) broth consisting of 9 g/l tryptone, 4.5 g/l
yeast extract, and 4.5 g/l NaCl was prepared along with 300 ml of LB broth consisting of
10g/l tryptone, 5 g/l yeast extract, and 5 g/l NaCl and 200 ml of glucose solution
consisting of 300 g/l glucose. All solutions were sterilized. After having been sterilized
and cooled, 100 ml of the 300 ml LB broth was inoculated with E. coli.
After allowing the inoculated LB to incubate at 37oC for 8 hours, the bacteria cells were
removed from the LB using a centrifuge. The cells were then re-suspended in 100 ml of
fresh LB from the 300 ml stock solution. 150 ml of glucose solution and the bacteria
suspension were added to the 700 ml of LB in the fermenter. Keeping the mixture stirred
and heated to 37oC, samples were taken every thirty to sixty minutes and the optical
density recorded. The cells were removed from 6 ml of each sample by centrifugation
and the liquid frozen and stored so that it could be analyzed by HPLC chromatography
later.
The fermentation was run until the optical density of three consecutive samples failed to
rise. At least six samples were collected from the exponential phase and linear phase
each.
Results and Discussion
We found that, for the three strains of E. coli under observation, the exponential growth
phase was short compared to the linear growth phase. Wild type E. coli exhibited an
exponential growth phase lasting only 1.5 hours, KO11 exhibited an exponential phase
lasting 2 hours, and LY01 grew exponentially for 3 hours.
These growth profiles were expected as KO11 was engineered to produce ethanol and so
should have a higher ethanol tolerance than wild type E. coli. LY01 inherently has a
higher ethanol tolerance than KO11, as LY01 is nothing more than KO11 that was
selected for high ethanol tolerance.
Specific rates were determined for the exponential growth phase of each of the three
bacteria. A plot of the natural logarithm of dry cell mass (in mg) against time was
generated. The slope of this plot is the specific rate. For wild type E. coli the specific
rate was 1.464, for KO11, it was 1.2524, and for LY01, it was 1.0378. The specific rates
for the exponential phases of each of the three bacteria were calculated using the
following equation.
 = d(ln(x))/dt
In this equation,  is the specific rate, x is dry cell mass, and t is time. The difference in
the specific rates of wild type E. coli and KO11 is most like due to the addition of
ethanol-producing genes and the removal of the succinate-producing gene. The lower
specific rate of LY01 is most likely due to the mutations undergone be KO11 as it
became more tolerant of ethanol. Graphs of the exponential phase for each of the strains
can be seen below.
Wild Type E. Coli
y = 1.464x - 3.8197
ln(cell mass)
R2 = 0.9986
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
-3.500
-4.000
-4.500
0.000
0.200
0.400
0.600
0.800
Time (hrs)
1.000
1.200
1.400
1.600
KO11
y = 1.2524x - 3.5625
R2 = 0.9921
ln(cell mass)
0
-1
-2
-3
-4
0.000
0.500
1.000
1.500
2.000
2.500
3.000
hours
y = 1.0378x - 3.4944
R2 = 0.9948
LY01
ln(cell mass)
0
-1
-2
-3
-4
0
0.5
1
1.5
2
2.5
3
3.5
hours
The amount of time spent in the linear growth phase also varied for each of the strains, as
did the rapidity of growth. Wild type E. coli grew the most rapidly in the linear phase
achieving a peak optical density of 9.23. KO11 grew more slowly in the linear phase and
achieved a peak optical density of 7.27. LY01 grew more slowly in the linear phase than
either of its two predecessors and achieved a peak optical density of 6.15.
Specific growth rates for the linear phase can be found by dividing the slope of the cell
mass versus time plot by the cell mass at any given point. Cell mass can be determined
from optical density by the following equation.
A = 0.0005 * B
In this equation, A is dry cell mass in mg and B is optical density. This equation is a
result of the experiment outlined in appendix I. Since the slope of the growth plot is
constant and the dry cell mass is always increasing, the specific rate in the linear phase is
always decreasing. Graphs of the linear growth phase for each of the strains can be seen
below.
O.D.
Wild Type E. coli
8.000
7.000
6.000
5.000
y = 1.4459x - 3.1945
R2 = 0.9978
4.000
3.000
2.000
1.000
0.000
4.000
4.500
5.000
5.500
6.000
6.500
7.000
7.500
Tim e (hrs)
KO11
7.000
6.000
y = 1.3883x - 3.0437
R2 = 0.9985
O.D.
5.000
4.000
3.000
2.000
1.000
0.000
4.000
4.500
5.000
5.500
6.000
6.500
7.000
Time (hrs)
O.D.
LY01
5.000
y = 0.4058x + 0.1234
4.500
R2 = 0.9961
4.000
3.500
3.000
2.500
2.000
1.500
1.000
0.500
0.000
7.000
8.000
9.000
10.000
11.000
Time (hrs)
The concentration of metabolic products formed by these three strains varied. Wild type
E. coli produced primarily lactate while KO11 and LY01 both produced mainly ethanol.
In KO11 and LY01 the concentration of succinate was essentially zero. The final
concentration in g/l of ethanol was 4.2 for wild type E. coli, 18.3 for KO11, and 12.1 for
LY01. These results agree with expectations. KO11 and LY01 should not have
produced any succinate, as the pathway leading to the formation of succinate is turned off
in these two bacteria. The final ethanol concentration in LY01 is lower than KO11. A
flux analysis will be necessary to determine why this is true.
The concentrations of formate, acetate, and lactate, were between 0 and 10 g/l with the
exception of lactate in wild type E. coli, in which it was the major product. Graphs
depicting the metabolic products formed by each of the three bacteria are shown below.
Wild Type E. coli Organic Acids
25
20
Succinate
Lactate
g/l
15
Formate
10
Acetate
5
Ethanol
0
0.00
2.00
4.00
6.00
8.00
10.00
12.00
hours
KO11 Organic Acids
g/l
20
succinate
15
lactate
10
f ormate
5
acetate
ethanol
0
0.000
3.000
6.000
9.000
12.000
hours
LY01 Organic Acids
15
succinate
g/l
10
lactate
5
formate
0
acetate
0.0
5.0
10.0
15.0
hours
20.0
25.0
30.0
ethanol
Conclusions
While an analysis of the metabolic fluxes will be necessary for a complete understanding
of these bacteria, we were able to draw some conclusions from what we have done thus
far. We have found that wild type E. coli grew faster than the other two and reached a
higher optical density. The genetic modifications necessary to produce the KO11 strain
resulted in a slower specific growth rate as well as the expected increase in ethanol
production. LY01 grew more slowly than the other two and reached the lowest optical
density of the three. It produced less ethanol than KO11 but more organic acids.
The next step in this project will be to complete a flux analysis. This will enable us to
answer some lingering questions regarding the decrease in ethanol production from
KO11 to LY01 and the increase in the production of organic acids between these two
strains.
Acknowledgements
This research was done at Iowa State University by the research groups of Dr. Jacqueline
Shanks and Dr. Ramon Gonzolaz. This material is based upon work supported by the
National Science Foundation under Grant EEC-0087696.