THE EFFECT OF PH ON THE GROWTH OF ESCHERICHIA COLI IN A LURIA-BERTANI BROTH ENVIRONMENT

Kavin Suhirtharen

Illinois Mathematics and Science Academy

ABSTRACT

This study was done with the purpose of testing whether different pH levels would affect the growth of E.coli. In order to perform the experiment, 10 test tubes for each pH level being tested (5 to 9) were each filled with 10 mL of Luria-Bertani broth and 150 μL of E.coli. Each test tube was then tuned to its assigned pH level using NaOH & HCl. After 48 hours of growth in a 32°C incubator, the test tubes were all taken out and their light absorbances were taken using a photospectrometer. A one-way five sample ANOVA test on the absorbances of each pH level told us that there was a significant difference between the means of the samples. From the results of the experiment, it was concluded that pH level significantly affects E.coli growth. It was also concluded that growth rates of E.coli rise as its environment’s pH rises, but only up until the environment peaks at a pH of 8; after its environment is any higher than a pH of 8, the growth rates start to decrease again.        

INTRODUCTION

Escherichia Coli is a prokaryotic, gram-negative, anaerobic bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (Blount, 2015). It is most known for its hardy, non-pathogenic, and versatile strains that grow on many different nutrients, as well as its limitless applications in the real-world (Lim, Yoon, & Hovde, 2010). However, even though E.coli is very versatile, it is hard to grow at a consistent rate for experimental purposes. Luria-Bertani (LB) broth is a rich bacterial medium used in lab environments for the cultivation and maintenance of recombinant strains of Escherichia Coli (Sezonov, Joseleau-Petit, & D'Ari, 2007). It is popular with bacteriologists because it permits fast growth and good growth yields for many species. Bacteria, especially E.coli, is affected by a variety of factors that can drastically change its growth. A factor that could be tested to see whether it significantly affects bacteria growth is pH level. Acidity and alkalinity are measured on the pH scale, which ranges from 1 to 14, with one being the most acidic, fourteen being the most alkaline, and seven being neutral (Cunningham, 2009). The purpose of this study was to determine whether various pH levels affect the growth of E.coli, and if so, at what pH levels E.coli thrives the best and at what pH levels E.coli growth is hindered. We also set out to determine whether E.coli growth could actually be controlled by changing its environment’s pH level. Many other studies have been conducted on this subject, but all have had varying, inconsistent results. If it is found that different pH levels affect the growth of E.coli, then the information could open new doors to research in fields such as food preservation and genetic engineering. 

METHODS AND MATERIALS

The growth of Escherichia coli in test tubes of different pH levels was monitored for a period of 48 hours. Ten test tubes of each pH level being tested (5, 6, 7, 8, and 9) were each set up with 10 mL of Luria-Bertani (LB) broth (this totaled to 50 test tubes). Each test tube was labeled with its assigned pH level and stored in a test tube rack whenever necessary. In order to adjust the pH level of each test tube to its assigned pH, NaOH was used to increase pH while HCl was used to decrease pH. Using a guess and check method, both of these chemicals were dropped into the test tubes using a micropipette and the pH of each test tube was measured using pH strips to ensure that every trial was accurate. Once each test tube had been set to its assigned pH level, a micropipette was used to drop 150 μL of E.coli into each tube (Figure 1). Secure caps were then placed onto each test tube to prevent any contamination and the tubes were put into an incubator of 32°C for 48 hours.

Figure 1. Example of how a micropipette was used to take E.coli and pipette it into the test tubes.

        After 48 hours of incubation, the E.coli test tubes were all taken out of the incubator and left outside for an hour to cool to room temperature. During this time, a photospectromoter was set up in order to collect the results (Figure 2). To be able to use the photospectrometer, it was connected to a computer that ran the software Vernier Spectral Analysis, which allowed us to view the absorbances of each E.coli trial. However, before data could start being collected, the photospectrometer had to be calibrated. To do this, a cuvette was filled up halfway with only LB broth and inserted into the photospectrometer (Figure 2). In Vernier Spectral Analysis, the wavelength was set to 600 nm and then the calibration occurred automatically. Once the photospectrometer was calibrated, it was ready to collect data from all 50 of the trials.

Figure 2. The photospectrometer that was used to collect data in our experiment. Note how the cuvette is properly aligned in the photospectrometer and the solution in the cuvette is only half-filled. 

        For each trial, a test tube was poured halfway into a cuvette and the cuvette was placed into the photospectrometer to have its absorbance read. This process continued on for the remainder of all 50 test tubes, and the data was logged into an Excel workbook (which was organized by pH level).  Finally, once all of the data had been collected for each test tube, the experiment was terminated and all of the data was analyzed.

        When analyzing the data, descriptive statistics in Microsoft Excel was run on each pH data set for values such as the sample variation, standard of error, and standard deviation. All of the trials for each pH level were averaged in order to get a single, accurate number that represented their trial values, respectively. In order to test whether the mean absorbances from each pH level were statistically different, a one-way five sample ANOVA test was run.

RESULTS

Table 1 shows the absorbance of each E.coli trial at every pH level that was tested (5-9). A higher absorbance suggests that there was more bacteria growth within the trial. Figure 3 exhibits a visual representation of the mean absorbances of the E.coli broth at each pH level tested. After running a one-way five sample ANOVA test on the data displayed in Table 1 (excluding the final row), it was concluded that there was a significant difference between the means of the five samples (F = 28.95, df = 4, p-value = <0.0001) (Figure 3).

Table 1. Absorbance of E.coli broth of ten trials in pH levels ranging from 5 to 9. Each trial was 10 mL and the results were collected after 48 hours of uninterrupted growth. Au = Absorbance units.

Figure 3. Mean absorbance of the E.coli broth for each pH level. After running the ten trials for each pH level, their averages were recorded and graphed above. The standard error bars represent plus or minus one standard deviation around the mean. Au = Absorbance units.

DISCUSSION

From our results, it was concluded that acidity/alkalinity(pH) does significantly affect the growth of E.coli (Table 1). This is proven by the results of the ANOVA test done on the data in Table 1, as we concluded from it that the means of all the different pH samples were statistically different from each other. From Figure 3, it was also concluded that an environment of pH 8 is the most supportive for E.coli growth, while an environment of pH 5 is the least supportive for growth (within the range of pH levels tested). Using this, it was deduced that growth rates of E.coli rise as its environment’s pH rises, but only up until the environment peaks at a pH of 8; after its environment is any higher than a pH of 8, the growth rates start to decrease again (Figure 3). These conclusions are significant because they tell us that E.coli growth can be controlled by changing its environment’s pH, which has numerous real-world applications in fields such as genetic engineering and food preservation (see paragraph three for more information). 

Our conclusions related to the conclusions formed by M. Benjamin and A. Datta when they conducted an experiment regarding the tolerance of E.coli in different pH environments. They concluded that E.coli has the highest rate of growth in environments of pH 7, 8, and 9 (Benjamin & Datta, 1995). Our results do support this conclusion as the mean absorbances from each sample in our experiment were highest in pH levels 7, 8, and 9 (Figure 3). However, our conclusions did not support the conclusions of N. Parhad and N. Rao, who conducted a similar experiment regarding the effect of pH level on the survival of Escherichia coli. After conducting their experiment, Parhad and Rao claimed that E.coli could not grow in environments that had a pH of 8.5 or greater (Parhad & Rao, 1974). From the results of our experiment, E.coli was still growing in the pH 9 environment since the mean absorbance of the pH 9 solution was 0.1926 Au (Table 1). As this number was greater than 0, there had to have been at least some bacteria growth within that pH environment. However, there could have been flaws within our experiment that caused us to get inaccurate results that were not similar to Parhad and Rao’s (see paragraph 4).

As a result of this experiment, there are several real-world applications that could benefit society. E. coli is optimal for lab experiments because of its hardiness, rapid-growth rate, and versatility (Blount, 2015). However, there have been situations where its growth has simply not been fast enough to keep up with developing technology. An example of this is in molecular cloning, which relies significantly on the speed of bacteria growth. The faster E.coli is able to grow, the faster it can be cloned (Olsvik, Wasteson, Lund, & Hornes, 1991). Since we now know that an environment of pH 8 supports bacteria growth the most, we can speed up the process of molecular cloning by having the E.coli grow in a similar environment. Another example of how controlling the pH level of the E.coli’s environment can benefit society is with food - specifically food preservation. E.coli grows at incredibly fast rates in food, and this is in part due to the neutral environment it thrives in (E. coli – the biotech bacterium, 2019). Since we have concluded that E.coli grows slower in lower pH levels, this problem could be solved by preserving food in environments that are more acidic. Research should continue to be done in this area to ensure that our results are accurate and to investigate other real-world applications that could come out of being able to control E.coli growth with acidity/alkalinity.

While we interpreted our results to conclude that pH level does have a significant effect on E.coli growth, we did not see any other interpretations/explanations from the results we collected. However, within Table 1, there were a few irregularities in the data set for each pH that could have affected our results. These could have been caused by, but are not limited to: human error (when measuring liquids or with keeping the lab environment sterile), the surrounding environment temperature, and the accuracy of the photospectrometer (it could have become miscalibrated at some point during the experiment). Furthermore, to test the pH level of each solution, we used pH strips, and since our eyes are not always color accurate, the pH solutions could have been slightly off of what they were intended to be.

Literature Cited

Benjamin, M. M., Datta, A. R. (1995). Acid tolerance of enterohemorrhagic Escherichia coli. American Society for Microbiology.  Retrieved from https://aem.asm.org/content/aem/61/4/1669.full.pdf

Blount, Z. D. (2015). The unexhausted potential of E. coli. ELife, 4. doi: 10.7554/elife.05826

Cunningham, E. (2009). What Impact Does pH Have on Food and Nutrition? Journal of the American Dietetic Association, 109(10), 1816. doi: 10.1016/j.jada.2009.08.028

E. coli – the biotech bacterium. (2019). Retrieved from https://www.sciencelearn.org.nz/resources/1899-e-coli-the-biotech-bacterium.

Lim, J. Y., Yoon, J., & Hovde, C. J. (2010). A brief overview of Escherichia coli O157:H7 and its plasmid O157. Journal of microbiology and biotechnology, 20(1), 5–14.

Olsvik, Ø., Wasteson, Y., Lund, A., & Hornes, E. (1991). Pathogenic Escherichia coli found in food. International Journal of Food Microbiology, 12(1), 103–113. doi: 10.1016/0168-1605(91)90051-p

Parhad, N., & Rao, N. (1974). Effect of pH on Survival of Escherichia coli. Journal (Water Pollution Control Federation), 46(5), 980-986. Retrieved from www.jstor.org/stable/25038739

Sezonov, G., Joseleau-Petit, D., & D'Ari, R. (2007). Escherichia coli physiology in Luria-Bertani broth. Journal of bacteriology, 189(23), 8746–8749. doi:10.1128/JB.01368-07

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