Acceleration of the Growth of
Escherichia coli
Group R1
Birche Fishback (TTK)
Rich Dela Rosa (Facilitator)
Tammy Mirensky (Presenter)
Lee Zeng (semi-Scribe)
Abstract
The primary goal of this experiment was to accelerate the growth rate of the K-12 strain
of Escherichia coli through the addition of nutrients to the media of the standard experiment (10.0
 0.1g bacto-tryptone and 5.0  0.1g yeast extract). [11] This was accomplished by the addition
of glucose, a carbon source, in the form of corn syrup to the control media while maintaining
constant environmental growth conditions. Additionally, using scientifically published paper [9]
a 35.1% increase in growth rate constant was predicted. One control and two experimental trials
were then performed. The growth rate constant for the control experiment was 0.0188 min-1 
4.05%. The average growth rate constant for the two experimental trials conducted was 0.0239 
min-1  6.05% thereby yielding a 27.13% increase in the growth rate constant from that of the
control experiment. This percentage increase deviated from the predicted increase by 22.7%.
Background
The growth rate of Escherichia coli is dependent upon the source and amount of carbon
in the media. The weight yield of microbial cells is directly proportional to the amount of the
energy source in the medium when this energy source is the growth-limiting factor [12]. Through
fueling reactions, the carbon source is broken down and simultaneously is assimilated into cell
substance, providing the precursor metabolites and energy as ATP or its equivalent [8]. For a
particular temperature, the specific growth rate depends upon the carbon source. The nature of the
dependence is strongly suspected that the rate of production of ATP must control the rate of
growth [6]. One biomedical application of accelerating the growth rate of bacteria would be to
grow genetically-engineered bacteria that produce beneficial enzymes that can be easily
extracted.
Carbohydrates and amino acids serve as potential sources of both carbon and energy [7].
The media used in the control and experimental trials was composed of yeast extract and bactotryptone, both of which provide carbon to the Escherichia coli in the form of amino acids and
carbohydrates [11]. To determine the amount of glucose added to the control media in order to
achieve an increase in the growth rate, it was first necessary to calculate the amount of carbon
present in the control media.
2
Using the assay of the yeast extract (Table 1A) and bacto-tryptone (Table 2A), the mass
of each amino acid was calculated in the 5.0  0.1g of yeast extract and 10.0  0.1g of bactotryptone used in the standard experiment. The mass of carbon contributed by each amino acid
was calculated by multiplying the percent of carbon present in each amino acid (molecular weight
of each amino acid and the molecular weight of the carbon present in each amino acid) by the
mass of the amino acid present. The mass of carbon contributed by each amino acid was summed
to obtain the total contribution of carbon from the amino acids. The carbon contributed by amino
acids in the 5.0  0.1g yeast extract was calculated to be 1.328g (Table 2A), and the carbon
contributed by the amino acids in 10.0  0.1g of bacto-tryptone was 3.696g (Table 3A).
Additional carbon is supplied to the Escherichia coli by the carbohydrates present in the
yeast extract and the bacto-tryptone. The yeast extract is composed of 15% carbohydrate, of
which 17.5% is carbon. This yielded an additional 0.131g carbon for the 5.0  0.1g of yeast
extract utilized in the media. Bacto-tryptone is composed of 7.7% carbohydrate of which 17.5%
is carbon. Therefore, there is an additional 0.135g carbon for the 10.0  0.1g of bacto-tryptone
present in the standard experiment.
The total carbon contributed to the Escherichia coli by the amino acids and carbohydrates
(5.289g carbon) in the control trial was used in the determination of the amount of carbon that
was added to the media in order accelerate the growth rate. This was based on a previous
experiment conducted by Paalme et al in which the K-12 strain of Escherichia coli was grown in
media of different carbon sources [9]. The maximum growth rate for Escherichia coli observed
by Paalme was achieved when using a media composed of casamino acids and glucose as carbon
sources. In Paalme’s experiment, the addition of 3.5g glucose to a media containing 1.5g
casamino acids (composed of the same amino acids as the bacto-tryptone and yeast extract)
increased the growth rate of the K-12 strain of Escherichia coli from 0.57 h-1 to 0.77 h-1, which
was a 35.1% increase [9]. Using the assay of casamino acids, the amount of carbon contributed
by 1.5g casamino acid was calculated to be 0.513g carbon (Table 3A).
The ratio of the mass of carbon from the non-glucose source to the mass of glucose added
to achieve the maximum growth rate in Paalme’s article was used to determine the amount of
glucose that was added to the control experiment to accelerate growth. The mass of carbon from
the non-glucose source (bacto-tryptone and yeast extract) for the standard experiment was
determined to be 5.289g carbon. Therefore the ratio from the literature values (0.513g carbon
from casamino acids/ 3.5g glucose) yielding the maximum growth rate was used to determine the
amount of glucose added to achieve an increase in the growth rate for this experimental
procedure. For this experiment, the mass of carbon from the non-glucose source corresponded to
3
the addition of 36.09g glucose. The glucose was added in the form of corn syrup to ensure the
sterility of the media. Corn syrup is 75% glucose by weight [2]. Therefore, 36.09g glucose
corresponded to the addition of 48.12g corn syrup.
Bacteria experience several different phases of growth, including the lag phase, log
phase, stationary phase, and death phase. In assessing the growth rate constant, only the log
phase was relevant. The exponential growth rate experienced during this phase corresponds to
the growth rate of the Escherichia coli. Through the addition of 3.5g glucose to the growth media
containing 1.5g casamino acids, Paalme obtained a 35.1% increase in the growth rate constant.
Using this value, it was expected that when following the experimental procedure of Paalme, the
growth rate constant for the experimental trials conducted (through the addition of glucose to a
non-glucose carbon source using the same ratio determined from Paalme’s experiment) would
have increased from 0.0188 min-1 in the control trial to 0.0254 min-1.
Materials

PennCell Culture Apparatus

Milton-Roy Spectonic 20D Spectrophotometer

All American Electric Pressure Steam Sterilizer Model 25X

Assorted glassware and plastic ware (2 cuvettes)

E. Coli Microkwik cultures

Thermometer

pH meter

10.0  0.1g bacto-tryptone

5.0  0.1g yeast extract

10.0  0.1g sodium chloride

corn syrup (glucose source)

de-ionized water

NaOH

parafilm
4
Procedure
The procedure for carrying out this experiment entailed the same basic outline as the
procedure used in experiment 1 [11]. The environmental conditions were initially set to a pH of
7.00  0.02, a temperature of 38.00  0.01°C, and an airflow of 0.75. This set of conditions
ensured an optimal growing environment for the bacteria [3]. The PennCell Apparatus was
calibrated with 1000.00  0.01g water, which has a volume of 1000.00  0.01mL. In the control
trial, 1000.00  0.01mL media (specifically 10.0  0.1g bacto-tryptone, 5.0  0.1g yeast extract,
10.0  0.1g sodium chloride, and 1000.00  0.01g de-ionized water) was used. One control trial
was conducted in which a base-line growth rate of the Escherichia coli was determined. Two
identical experimental trials were completed throughout the subsequent weeks. The experimental
procedures differed from the controlled protocol by the addition of 48.12  0.01g corn syrup to
950.00  0.01mL control media. The total volume of media, with the corn syrup, was brought up
to 1000.00  0.01mL but the addition of water to the calibration line of the Penn-Cell Apparatus.
The growth rate of the Escherichia coli was determined by the graph of the natural log of
absorbance as a function of increasing time. Absorbance measurements were taken using the
spectrophotometer. During the experimental trials, the high growth rate of the Escherichia coli
resulted in the increased absorbance of the sample to the extent that the maximum reading range
of the spectrophotometer was surpassed. Because the absorbance values did not fall within the
working range of the spectrophotometer, it was necessary to dilute the samples so that readings
could be taken accordingly. This problem was not anticipated in designing the experimental
procedure so that supplementary media was not prepared for proper dilution. Therefore, all of the
media samples (from the point of exceeding the working range of the spectrophotometer on) were
diluted with water so that the samples were within the reading range of the spectrophotometer.
All dilutions were conducted by the addition of 2 parts water to one part sample. To reduce
experimental error and to remain consistent throughout the entire trial, the same micropipette was
used throughout the entire trial for all of the dilutions. In addition, it was important that all of the
samples be diluted in the same manner to guarantee the same shift throughout the entire growth
rate curve. In doing so, the slope of the log phase, which is equivalent to the growth rate
constant, in the graph of ln absorbance as a function of time was determined.
Readings were initially taken every 20 minutes during the lag phase of growth. The time
interval between readings was decreased to approximately 10-15 minutes during the log phase to
ensure ample data points for data analysis. Once the linear growth phase was attained,
5
subsequent readings were conducted for approximately 3 hours. To ensure that the samples
obtained were representative of the entire growth media and the actively growing bacteria, the
residual sample remaining in the glass tube after each reading was returned to the growth media
and fresh sample was taken. In addition, all samples were conduced in the same cuvettes to
reduce experimental error.
To ensure optimal growing conditions, the pH of the growth media was regulated
throughout all trials, both control, as well as experimental. The pH was measured using a pH
meter every 30 minutes. Upon growth of the bacteria, the pH was found to decrease as acid was
produced. The levels of acidity decreased and the pH brought back up to the initial desired
condition using NaOH to neutralize the media. The NaOH was added drop-wise to the growth
media in the PennCell Apparatus. In doing so, care was taken to ensure that the NaOH was
placed in the growth media and not on the foam sitting above the growth media by inserting the
pipette directly into the media.
Results
0.5
ln Absorbance
0
-0.5100
150
200
250
300
350
400
-1
-1.5
y = 0.0188x - 6.8518
-2
-2.5
-3
-3.5
-4
Time (minutes)
Figure 1: Log phase of bacteria represented in the graph of ln Absorbance vs. time for the
control trial performed. In this trial, the media consisted solely of bacto-tryptone, yeast
extract, sodium chloride, and water. The growth rate was determined to be 0.0188 min-1.
6
ln Absorbance
0.5
0
220
-0.5
-1
270
320
370
y = 0.0244x - 8.6881
-1.5
-2
-2.5
-3
Time (minutes)
Figure 2: Ln Absorbance vs. time for the log phase of bacteria growth for the first experimental trial
performed. In this trial, the media consisted of that for the control with the addition of 48.12  0.01g
corn syrup. The growth rate was determined to be 0.0244 min-1.
ln Absorbance
0.5
0
200
-0.5
220
240
260
280
300
320
-1
y = 0.0234x - 6.8519
-1.5
-2
-2.5
Time (minutes)
Figure 3: Ln Absorbance vs. time for the log phase of bacteria growth for the second experimental
trial performed. In this trial, the media consisted of that for the control with the addition of 48.12 
0.01g corn syrup. The growth rate was determined to be 0.0234 min -1, the slope of the regression.
7
Control
Experimental 1
Experimental 2
Predicted (35.1%
increase)
Growth rate
constant
0.018755
0.024355
0.023357
0.025338
lower 95% limit Upper 95% limit
0.017994
0.023673
0.022147
0.019515
0.025037
0.024566
Table 1: Growth rate constants and their 95% confidence interval for the control and two
experimental trials. The predicted 35.1% increase in growth rate constant assumes the control
growth rate constant as exact.
Discussion
From the complete plots of ln absorbance versus time, only the linear portion, which
represented the log phase, was analyzed. Regressions were performed using various
combinations of data points on this linear region. To ensure that the data points were
representative of the log phase, points near the lag phase or stationary phase were not considered
in any combination of data points. The set of maximum data points with an R2 value closest to
1.000 was chosen as the representative regression whose slope was equivalent to the growth rate
constant.
As can be seen from Figure 1, bacteria growing in media consisting solely of bactotryptone and yeast extract (control trial) grows at a rate of 0.0188 min-1 with an upper 95%
confidence limit of 0.0195 min-1 and a lower 95% confidence limit of 0.0180 min-1. Through the
addition of 48.12  0.01g corn syrup (36.09g glucose), the growth rate increased to an average of
0.0239  3.95% min-1, thereby showing a 27.13%  5.04% increase in the growth rate. The slope
of the in Figure 2 yielded a growth rate of 0.0244 min-1 with upper 95% confidence limit of
0.0250 min-1 and lower 95% confidence limit of 0.0237 min-1 for the first experimental trial. The
second experimental trial, in which the growth conditions remained the same as those for the first
experimental trial, yielded a growth rate of 0.0234 min-1 with upper 95% confidence interval
0.0246 min-1 and lower 95% confidence interval 0.0221 min-1, as can be seen in Figure 3.
8
Figure 4: Graphical representation of growth rate constant values with their 95% confidence
intervals including the predicted 35.1% growth rate constant increase from the control.
As shown in Figure 4, the lower bound 95% confidence limit of either experimental
growth rate constant does not overlap with the upper bound 95% confidence limit of the control
growth rate constant. This indicates that it is 95% probable that the increase in growth rate
constant was not due to experimental error. It is also worth noting that the confidence intervals of
the two experimental trials overlapped with each other thereby indicating reproducible results.
However, because only two experimental trials were performed, the 95% confidence interval of
the mean was 0.006340 min-1. To obtain more precise experimental results, the confidence
intervals must be smaller. This can be accomplished by performing more than two experimental
trials. In addition, the second experimental growth rate constant had the value for the predicted
increase constant lie in its upper bound 95% confidence limit. Therefore, this set of data indicates
that the predicted growth rate falls within the experimental results for this trial.
The growth rate constant does not solely depend on the number of carbons in its energy
source, but also on the actual type of carbon source. In the assays of bacto-tryptone, yeast
extract, and casamino acids, it can be noted that they are all composed of the same amino acids.
Therefore, the carbon added in the experiment was comparable to that used by Paalme because
the carbon was derived from the same sources. Though other sugars may have the same number
of carbons as glucose does, there may be a different efficacy in the transportation of their carbons
to the bacteria for use as an energy source. If the experiment performed by Paalme et al used a
different carbon source other than glucose to achieve their maximum growth rate, the trials in this
experiment must have been performed with that same source to achieve comparable results.
9
Paalme’s experiment differed from what was performed in this laboratory with his use of
the a-stat [9] method of growing his culture. This technique included a continuous addition of an
energy source to the media while taking out a certain percentage of the bacteria. This prolonged
the log phase of the bacteria growth and also allowed for the amount of accessible carbon in the
media to remain constant thus allowing a greater amount of data to be analyzed. In contrast, the
procedure employed in this lab called for a set amount of bacteria growing in a practically
unchanging volume of media, ultimately exhausting the energy source. The two differing
methods in these labs, however, are comparable because the aspect being examined is the
percentage increase in the growth rate constant rather than the contrasting of numerical values of
the growth rate constants.
Another aspect of this project, which could be utilized in future trials and is indirectly
another means of observing bacteria growth, is pH measurement and manipulation. Escherichia
coli produce lactic and formic acid as byproducts of growth, which lower the pH in a growth
media. In this series of experiments, 1M NaOH was added every 30 minutes in varying amounts
to neutralize the acidity of the media. This was done because the optimal pH for the growth of
the bacteria is neutrality. In addition, the growth rate was determined based on the media rather
than on the alteration of environmental conditions (i.e. pH). The relationship that could be
exploited in future experiments is the concentration of bacteria’s relationship to the production of
acid, and thus pH, of the media. An increasing amount of bacteria results in a greater amount of
acid released into the media, thus lowering the pH by a measurable amount. The assumption here
is that the amount of NaOH added to increase the pH of the media to 7 is proportional to the
amount of acid produced by the bacteria, and therefore is proportional to the amount of bacteria
in the media. For a modified project, the NaOH necessary to maintain a neutral pH can be
recorded to create a function relating the amount of bacteria present to the NaOH added. Special
care would have to be taken, however, to add a precise amount of base that results in a media
with a pH of exactly 7. Difficulties could result due to the fact that time is needed for the base to
equilibrate with the system. A precise determination of the relationship could be difficult to
obtain due to this fact.
10
Appendix
%amino acid
ALANINE
4.47
ARGININE
3.16
ASPARTIC ACID 6.32
CYSTINE
0.513
GLYCINE
2.83
GLUTAMIC ACID 11.42
HISTIDINE
1.25
ISOLEUCINE
3.07
LEUCINE
4.48
LYSINE
4.75
METHIONINE
0.944
PHENYLALANINE 2.36
PROLINE
2.44
SERINE
3
THREONINE
2.87
TRYPTOPHAN
0.99
TYROSINE
0.801
VALINE
3.88
TOTAL
Mass of carbon in our sample of amino acid (g)**
0.090385808
0.043576073
0.114055323
0.010171085
0.045279246
0.233068749
0.029028906
0.098390024
0.123058271
0.116282866
0.018997359
0.077218246
0.063638582
0.051426736
0.057877048
0.032022668
0.023894061
0.099450875
1.327821927
Table 2A: Assay of amino acids in the yeast extract.
*All masses are based on the 5.0  0.1g of yeast extract that will be used in the experiment.
11
%amino acid
ALANINE
2.86
ARGININE
3.03
ASPARTIC ACID 6.11
CYSTINE
0.42
GLYCINE
1.75
GLUTAMIC ACID 17.05
HISTIDINE
2.02
ISOLEUCINE
4.4
LEUCINE
7.11
LYSINE
6.7
METHIONINE
2.57
PHENYLALANINE 3.71
PROLINE
7.45
SERINE
4.29
THREONINE
3.58
TRYPTOPHAN
0.71
TYROSINE
1.42
VALINE
5
TOTAL
Mass of carbon in our sample of amino acid (g) **
0.115661481
0.083566774
0.220531019
0.016654407
0.055999068
0.695940835
0.093821423
0.282030037
0.390600137
0.328040084
0.103439012
0.242779399
0.388612655
0.147080466
0.144390128
0.045931504
0.08471802
0.256316688
3.696113138
Table 3A: Assay of amino acids in the bacto-tryptone.
*All masses are based on the 10  0.1g bacto-tryptone used in this experiment.
12
%amino acid
ALANINE
3.26
ARGININE
2.2
ASPARTIC ACID
4.76
CYSTINE
0.16
GLUTAMIC ACID 15.3
GLYCINE
1.31
HISTIDINE
1.66
ISOLEUCINE
3.34
LEUCINE
5.47
LYSINE
5.71
METHIONINE
1.28
PHENYLALANINE 2.11
PROLINE
6.17
SERINE
2.19
THREONINE
2.41
TRYPTOPHAN
0.01
TYROSINE
0.47
VALINE
4.3
TOTAL
mass of carbon in 1.5 g casamino acid
0.019775687
0.009101332
0.025770728
0.00095168
0.183596943
0.003208267
0.011565116
0.032112966
0.045075585
0.041935273
0.007727739
0.020711504
0.048276646
0.011262455
0.014580176
9.70384E-05
0.004206071
0.033064853
0.51302
Table 4A: Assay of amino acids in casamino acids.
*All masses are based on 1.5g casamino acid used.
13
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14