The Effects of Carbon on E. Coli Cultivation
Group W1
April 30, 1998
Mukta Agrawal
Jessica Barag
Eric Brahin
Dominic Mangiardi
ABSTRACT
The specific aim of our project was to determine if the sugar glucose was an acceptable carbon donor for the growth of the bacteria E. Coli. This was performed by gradually eliminating all other sources of carbon in the bacteria’s growth medium, and analyzing the growth constant of the bacteria for each case. The first trial used a medium consisting entirely of Bacto-Tryptone, and had a growth constant of 0.0174 +/- .00205 1/min. The second trial used a medium of half Bacto-Tryptone and half glucose solution, and had a growth constant of 0.0139 +/- 0.00700 1/min. The final trial used a medium consisting entirely of the glucose solution, and had a corresponding growth constant of 0.0116 +/- 0.0015 1/min. We determined from the gradual decline in the growth constants that although glucose is an acceptable source of carbon for the bacteria, E. Coli growth is more effective in a solution that also contains amino acids and other minerals, which the Bacto-Tryptone provided. We also discovered that not only does the presence of amino acids and minerals in the medium propagate faster growth, it also provides for a higher total concentration.
BACKGROUND
E.Coli is a bacterium that is crucial to the biological sciences. It is often referred to as the “white laboratory rat” for the molecular sciences. More specifically, E. Coli is known as a chemoautotrophic proteobacteria, which means the cells consume organic molecules for both energy and carbon (1). In humans, they are found abundantly in the colon, and they feed on organic material that would otherwise be eliminated in feces. Their by-products are gases and vitamins, such as vitamin K, which is absorbed by the host. E. Coli is also a common source of plasmids for gene cloning. Although found in the body, certain strains of E. Coli can be very dangerous if it is ingested into the body. The bacterium has been found in some fast food restaurant’s hamburger meat, which caused food poisoning for many people (4). Thus, understanding the chemical kinetics of this organism is of great importance to bioengineers and many other scientists.
“For Microbes, growth is their most essential response to their physiochemical environment (3).”A bacterial population, such as E. Coli, follows a characteristic growth curves defined by four phases: the lag phase, the log or exponential phase, the stationary phase, and the death phase.
- Lag Phase: During this phase, the cell number remains constant. The bacteria are preparing for reproduction by synthesizing DNA as well as inducible enzymes needed for cell division.
- Log (or Exponential) Phase: This phase begins with the lag phase, and is characterized by a rapid increase in cell number. Over the course of this growth, the logarithm of E. Coli biomass linearly increases with time such that for a given interval, the number of E. Coli cells is directly related to the biomass of bacteria present.
- Stationary Phase: This stage occurs when the number of bacteria has reached a maximum upper bound. It implies that the growth rate is equivalent to the death rate. This appears as a plateau on the graph of absorbance vs. time.
Death Phase: Eventually the number of live E. Coli cells begin to decline. This indicates the beginning of the death phase. There is no further division of cells, and the kinetics are often similar to the exponential growth period.
Figure 1: Growth stages of E. Coli
The four stages of bacteria growth as explained above (4).
During the exponential or logarithmic phase, the E. Coli cells replicate by binary fission, the process through which a cell divides to form two identical cells. These two cells then both undergo binary fission themselves, forming a total of four cells, then again to form eight, and continue to multiply in this exponential manner. More scientifically, the growth rate of E. Coli can be characterized by the Integrated First-Order Rate Law, which demonstrates that the concentrations of species in a reaction in a stable state depend on time (2). The mathematical definition is below:
ln[C]=-kt +ln[C0] (Eq. 1)(3)
In this equation, C is concentration, t is time, k is the growth constant dependent on the conditions, and Co is concentration at time t = 0. It demonstrates the linear relationship between time and the natural log of the concentration. Therefore, the slope of the straight-line graph would be the growth constant k. There are a few factors that affect the growth constant of E. Coli that must be held constant, such as temperature, pH, and air flow.
Picture 1: A rendering of the E. Coli bacterium (6) Picture 2: E. Coli under a microscope (7)
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However, for growth to even occur, the environment must provide the bacteria with nutrients. More specifically, microorganisms need a hydrogen donor, a hydrogen acceptor, a carbon source, a nitrogen source, and minerals. The growth medium needs to supply all of these factors.
APPARATUS AND MATERIALS
· Penn-Cell Culture Apparatus
-Sampling Apparatus
-Temperature Probe
-Air stone
-Dataplate digital hot plate/ stirrer
-Heating coil
- Gelman sterile filter (0.2 mm)
-Second Nature Whisper 700 air pump
-Magnetic stirrer
· Milton-Roy Spectronic 20D Spectrophotometer with cuvettes and cuvette holder
· NAPCO Scientific Co. Model 9000-D Autoclave
· Imperial II Radiant Heat Oven
· Stopwatch
· pH meter with 7 standard
· Eye dropper
· Assorted glassware and plastic ware
· E. Coli Microkwik cultures, Rehydrated Sterile Growth Solution and water
-Deionized water
-Bacto-Trypone
-Glucose
-Yeast extract
-Sodium Chloride
-5N NaOH
-5N HCl
PROCEDURE
The experiment was accomplished the same way the Chemical Kinetics and Cell Growth experiment from the BE 210 Laboratory Manual was performed. The same procedure was followed with the exception that it was performed three different times using three different medias. The media preparation was done two days before the trial was to be performed. The day before the experiment, the dehydrated E. Coli cells were rehydrated. On the day of the experiment (which started around 10 o’clock), the Penn-Cell apparatus was assembled and the media was added. When the temperature was at 37o C, the pH was 7.2, and the airflow rate was 0.75, the cells were added to the media. After approximately one hour the absorbance readings were taken, and they were read every fifteen minutes until 6 o’clock. A crucial part of the experiment was to monitor the pH every hour to make sure it did not drop below 7.2.
Since the purpose of our experiment was to see if Glucose could be the sole carbon (or sugar) source in the growth of E. Coli, the media would have to be altered. However, everything but the sugar source was controlled. These components consisted of 1000 mL of deionized water, 5 grams of Yeast Extract to ensure the cells get the needed vitamins, 10 g of Sodium Chloride to maintain a reasonable osmotic pressure, and the necessary amounts of Sodium Chloride and Hydrochloric Acid to maintain a pH of 7.2.
In the first week of the experiment, the carbon source consisted of 10 grams of Bacto-Tryptone, which is a form of Casein. Casein is a milk derivative that is obtained when acid is added to milk; the precipitate that forms is the Casein. The milk sugar, which is Lactose, remains at the in the liquid left behind and is refered to as whey. However, when the casein is filtered and separated, some Lactose is still on the precipitate. 7.7% of the Bacto-Tryptone is sugar, which leaves the rest to be amino acids.
In the second week of the experiment, the sugar source was half Bacto-Tryptone (5 grams) and half Glucose (0.385 grams). The way this was calculated was that the ratio of sugar sources was keep equal, and since Bacto-Tryptone is 7.7% sugar, it could be easily calculated. The third week of the experiment, the sugar source was completely Glucose. In this case, twice the amount of Glucose used in the week before (0.77 grams) was used.
RESULTS
After the completion of the three trials with the different growth mediums, we plotted three graphs of concentration vs. time. The graphs are shown in Appendix A. The data for the graphs may be found in Appendix B.
By using the Integrated First-Order Rate Law, one can find the growth constants of each week. The data used comes only from the section of the graphs that explicitly demonstrate the exponential phase equations. The domain chosen for the results was determined to be from three data points in to the experiment (lag phase) to the point of inflection of the line, which is the end of the exponential phase. The growth constants for each trial are shown below.
Table 1: Growth Constants
Week # / Medium / Growth Constant(1/min) / 95% limits / Doubling time
1 / Bacto-Tryptone / .0174 / ± .00205 / 39.8 minutes
2 / ½ Bacto-Tryptone
½ glucose / .0139 / ± .00700 / 49.9 minutes
3 / Glucose / .0116 / ± .00150 / 59.7 minutes
Inspection of the table shows that the growth constant decreases with each week, the second week’s constant declines by 20.1%, and the third week’s by 33.3%. This corresponds to an increase in the doubling time for each successive trial. Even thought the growth constants are different for each week, we wanted to ensure that they were statistically different, i.e. that the difference between them was greater than the error associated with the calculations. With the aid of Microsoft Excel, regression statistics were performed for each week to obtain the 95% confidence limits, which are shown in table 1. One can see that none of the intervals for any of the trials overlap, indicating that all of the growth constants are statistically different.
Another way to analyze the results is to look at the behavior of the cultures in equal time periods, from 45 – 105 minutes. Each graph is well into the exponential phase during this interval. Table 2 shows the number of cells at the ends of the interval and DCx, which is the change in cell concentration.
Table 2: The Change In Cell Concentration Over Time
Week # / Cx (45 mins.) / Cx (105 mins) / DCx1 / 1.37*10^7 / 4.77*10^7 / 3.40*10^7
2 / 2.28*10^7 / 4.75*10^7 / 2.47*10^7
3 / 1.46*10^7 / 3.00*10^7 / 1.54*10^7
During this time frame, as would be expected, the number of cells produced decreases with each week. The second and third week experiences a decrease in the production of bacterial by 40.4% and 54.7%, respectively.
DISCUSSION
The purpose of this experiment was to determine the effect of altering the carbon source while keeping the carbon concentration constant. There were two possible explanations for the change in the growth constant over the three weeks:
1. One of the sugars was more easily broken down than the other, or
2. The presence of amino acids affects E. Coli growth.
If one of the sugars was more easy to break down the other, than that would mean that the E. Coli did not have enough energy to break down the more complex sugar. It would not be a test of whether or not one sugar was a suitable carbon source. Before this experiment was performed, it was imperative to determine whether glucose could thermodynamically be a fair substitution for lactose. This was done by comparing the values of the DHc for each sugar. If the values were very different for the two sugars, it would imply that one is easier to break down than the other. This means that the E. Coli would have to do more work to obtain one sugar that the other. Lactose has a DHc value of 1350 calories/kg, and glucose has a DHc value of 669.94 calories/kg. Since lactose has a molecular weight of approximately twice that of glucose, and therefore twice the number of carbon atoms per molecule, they have roughly the same DHc value per carbon atom. Therefore it is safe to say that it will not affect the outcome of the experiment.
Because the variable was not the carbon concentration, it was concluded that the variable must have been the amino acid concentration, which decreased each week as the glucose was substituted for the Bacto-Tryptone. The Bacto-Tryptone has many amino acids added to it, which are listed in Appendix C. The yeast also contained amino acids, but since the same amount was added each week, it was not a variable. Therefore the decrease in the growth constant may be attributed to the lack of amino acids.
If this is correct, than the E. Coli should not have grown at all during the third week. However, the growth is explained by the conclusion that the bacteria derived the minimum necessary amino acids from the yeast, which accounts for the decline in growth rate for the three weeks.
There are two aspects of the results that warrant discussion. The first is the fact that the growth constant dropped each week. Although we determined that glucose could be used as a carbon source, which is demonstrated by the growth in the third trial, we also determined that glucose was not as effective as the Bacto-Tryptone. This can be easily explained. The components of Bacto-Tryptone are shown in Appendix C. It is immediately obvious that the Bacto-Tryptone contains many other substances besides carbohydrate. The presence of the multiple amino acids and minerals clearly provided for faster growth, as the growth constants supported.
There is another aspect of the growth curves, which could possibly be more important in determining the effectiveness of glucose as a carbon source. Appendix B shows the concentrations of the three trials with respect to time. It is immediately obvious that trials one and two reached a significantly higher concentration than trial three. This is important because glucose not only lowers the growth constant, it significantly lowers the total concentration that will be reached. This is an important fact in consideration of glucose as a media solution. Again, this can be attributed to the presence of amino acids and minerals in the Bacto-Tryptone solution, which were present in the first two trials but not the third.