We sought to find Rockstar’s impact on E.coli’s growth rate and generation time. Our hypothesis was that E.coli’s growth rate would be negatively impacted by Rockstar. In order to test our hypothesis, we performed serial dilutions, spectrometry analysis, and plating of a highly concentrated E.coli solution to relate absorbance to E.coli density per ml. Then we performed two replicate trials of 6 groups of varying concentrations (0-25%) of Rockstar and took spectrometry absorbance every 30 minutes until the control reached the end of the log phase. Using the standard curve we converted absorbance data to density and calculated growth rate and generation time. Each replicate trial produced extremely close data and we were able to determine that Rockstar negatively impacts E.coli’s growth rate (by 1.5% from control per 1% Rockstar) and positively impacts doubling time (by 2.4% from control per 1% Rockstar).
For our experiment, we decided to use Escherichia coli (E.coli). We wanted to investigate how the growth of E.coli would be affected by increasing concentrations of Rockstar energy drink being added to the growth medium. We wanted to use Rockstar as it was an energy drink with one of the lowest pH and we wanted to see how the E.coli’s growth would be affected by such acidic conditions as it is a known gut bacteria found in the lower intestine of warm-blooded organisms. E.coli is known to be able to cause disease in the human intestine tract. However, in order to do this, it must combat the acidic environments during the process of invasion. So, with pH values as low as 1.5 - 2.5 the stomach has been recognized as a natural antibiotic barrier. This is why E.coli finds more growth success in areas like the small intestine where it will encounter a much less acidic environment of around 4.0 - 6.0 pH (Hickey 1990). This relates back to why we chose Rockstar to put in our medium as its pH is approximately 2.53, meaning it should be able to mimic the antibiotic barrier of the stomach fairly well. Energy drinks in general, not just Rockstar, are known to cause adverse effects to our Gastrointestinal health and metabolic activities. First energy drinks contain very large amounts of sugar ranging anywhere from 21g to 34g per can (Gaul 2015). With all of this sugar, mainly in the form of high fructose corn syrup, high rates of energy drink intake may lead to the development of obesity or type 2 diabetes. In addition to this, many energy drinks may reduce the activity, diversity, and gene expression of intestinal bacteria, again leading to unhealthy changes in metabolic activity. The high amounts of caffeine present in these drinks can also decrease our body’s insulin sensitivity, explaining the rise in blood glucose levels after consumption documented in other studies (Alsunni 2015). In another review of how energy drinks affect the Gastrointestinal system, they look at the effects from the mouth down to the stomach and intestines. First, in the mouth, it is known that these energy drinks can cause increased relaxation of the esophageal sphincter and increased gastric acid secretion. Consequently, this then ties into them also being able to cause gastroesophageal reflux and gastric ulcers as a result in some cases of prolonged use. Next, the review moves to the GI tract; the glucose load from all the sugar in these drinks will delay or slow gastric emptying. This would be suboptimal in conditions of prolonged exercise or hot or humid conditions where hydration would be important for an individual. This trend of increased difficulty of hydration follows in the intestines as it is found that the greater the concentration of carbohydrates in a caffeine-containing energy drink, the slower the rate of water absorption. When the components of these drinks leave the small intestine they enter the large intestine and the colon where they may interfere with the gut microbiome, where trillions of bacteria and other microorganisms live in symbiosis. Alterations in diet, such as high energy drink consumption, can change the composition of the microbiome resulting in downstream changes in immune system function and or metabolism, for example, increases in fat deposition in the host. Now, one would need to consume very large amounts of these drinks on a regular basis in order to enact these changes and consequences, however, regular consumption will likely add to the insult on gut microflora imposed by our Western-style diet (Shearer 2014). In another study investigating how a variety of energy drinks in Saudi Arabia were affecting the growth of certain beneficial GI microorganisms, for example, Bifidobacterium and Lactobacillus spp, they found that in all of the twenty drinks tested bacterial population decreased by at least 4.0log CFU/mL. This indicates that more safety improvements to the contents of these drinks is needed as well as further consumer education on the impact that these certain ingredients may have on them (Aljaloud 2016).
Now we wanted to highlight how E.coli grows in humans as well as any acid or low pH resistance mechanisms it has that may come into play with our results for this experiment. It is known that E.coli has developed two-variable acidic stress response systems for different environmental pH ranges. For extreme acid stress, it has the acid resistance (AR) system response that includes 5 AR systems AR1-AR5 (Stincone 2011). For mild or moderate acid stress E.coli has the acid tolerance response (ATR) system. First, we will discuss the AR1-AR5 systems. The AR1 system is activated by an alternative sigma factor (RpoS) and a cAMP receptor protein (CRP), and due to the CRP’s involvement, the AR1 is repressed by glucose. Now, the AR2-AR5 systems are all dependent on extracellular amino acids and consist of antiporters and decarboxylase enzymes that are induced by a low pH environment and the extracellular amino acid. They offer E.coli acid resistance by consuming intracellular protons in the amino acid decarboxylation reaction to produce a less acidic internal pH for the given E.coli, using glutamate, arginine, lysine, and ornithine respectively; where among the AR systems, AR2 is by far the most effective as well as complex. All five of these AR systems have the ability to protect stationary phase E.coli cells from extreme acidity to prolong their survival. Only the AR2 and AR 3 systems are able to function during the exponential phase of bacterial growth. Now for the ATR system, which admittedly is not as well understood as the AR system(s). The ATR system is induced by exposing E.coli to moderate acid stress of around pH 4.5 - 5.8 and provides a very slight challenge to extreme acids, though not nearly as effective as the AR systems, of around pH 2.0 - 3.0. The ATR system is activated at mild pH’s in stationary phase cells by regulators RpoS and OmpR and activated in exponential phase cells by regulators Fur and PhoPQ; it is known that stationary phase cells are much more tolerant to acid than log-phase cells. Now that we have explained these complicated systems we can talk about the benefits E.coli has from them. With the AR and ATR systems, E.coli can survive, without growth, for several hours at pH as low as 2.0. It is known that the acid limit for E.coli growth is pH 4.0 in a rich medium so as E.coli passes through the stomach and enters the intestine it will transition from no growth to growth conditions (Xu 2020).
So for our experiment, our main question was if Rockstar energy drink would have an effect on the growth of E.coli, would it increase or decrease it? To answer this question we came up with a null hypothesis that Rockstar energy drink would have no effect on the growth or growth rate of E.coli. Consequently, our alternative hypothesis was that if the Rockstar energy drink had an effect on E.coli’s growth rate, it would decrease the growth rate. This question was interesting to us as we all had known that energy drinks, in general, are not good for us but did not know how it would actually affect us internally. We knew that these drinks had to be fairly acidic and that E.coli wouldn’t be able to grow in a super acidic environment so that is how we came to the hypothesis that we designed. To test this we concocted a series of solutions with increasing concentrations of Rockstar in the tryptic soy broth and E.coli mediums. The amount of Rockstar being increased by 5% in each solution from 0% up to 25% Rockstar. We then used the spectrophotometer at 585nm in order to measure turbidimetry where we measured this in the spectrophotometer every 30 min until the growth curves tapered off. We hoped to learn how a very acidic energy drink like Rockstar could affect a prominent bacteria’s growth and to tie this into how these drinks and overconsumption of them might be affecting one’s gut health.
E.coli was inoculated into tryptic soy broth and incubated at 37°C overnight until the sample became cloudy and nontransparent. Next, a serial dilution was performed down to a factor of 10^-7. The spectrophotometer was blanked with non-inoculated tryptic soy broth from the same batch, then absorbance values at 585nm were taken for the original solution and all dilutions. Then 0.1 ml of the 10^-7, 10^-6, 10^-5 dilutions were plated via the spread plate method. After 24 hours the plates were counted and back-calculated to determine the number of E.coli per ml in the original solution. Absorbance data was then used to calculate bacteria per ml by dividing the known bacteria per ml in the original solution by the recorded absorbance of the original solution, and then multiplying the absorbance at various dilutions.
E.coli was incubated at 37°C in tryptic soy broth overnight. Once this solution was mild yellow and very cloudy the sample was chilled in the refrigerator for a day. The Rockstar was opened and left to sit for 2 hours with a tinfoil cap. While the Rockstar sat the solutions for testing were prepared. Twelve 250 ml flasks were labeled A0-A5 and B0-B5. Varying concentrations of Rockstar and tryptic soy broth were added in order to reach 3% tryptic soy broth per flask and increasing concentrations of Rockstar by 5% (from 0-25%) in order to reach 100 ml of media (Table 1). Next, the spectrophotometer was blanked with an empty cuvette at 585nm. In order to blank the media per solution 1 ml of each media was analyzed with the spectrophotometer. Absorbances were recorded as the baseline for that media. During that process, the E.coli solution was put back into the incubator at 37°C for 20 minutes before use. Once the media was sampled 1 ml of the E. coli solution was added to each 250 ml flask then immediately placed into the incubator at 37°C. Every 30-minute interval each sample was removed in the same order that they were originally ordered and absorbances were found on 1 ml of removed media, which was then discarded.
Table 1: Added Contents to Each Sample
| Tube Label | mL of DI | Grams of TSB | mL of Rockstar | Total Media | Concentration of TSB | % Rockstar | | --- | --- | --- | --- | --- | --- | --- | | A0 | 100 | 3 | 0 | 100 | 3.00% | 0.00% | | A1 | 95 | 3 | 5 | 100 | 3.00% | 5.00% | | A2 | 90 | 3 | 10 | 100 | 3.00% | 10.00% | | A3 | 85 | 3 | 15 | 100 | 3.00% | 15.00% | | A4 | 80 | 3 | 20 | 100 | 3.00% | 20.00% | | A5 | 75 | 3 | 25 | 100 | 3.00% | 25.00% | | B0 | 100 | 3 | 0 | 100 | 3.00% | 0.00% | | B1 | 95 | 3 | 5 | 100 | 3.00% | 5.00% | | B2 | 90 | 3 | 10 | 100 | 3.00% | 10.00% | | B3 | 85 | 3 | 15 | 100 | 3.00% | 15.00% | | B4 | 80 | 3 | 20 | 100 | 3.00% | 20.00% | | B5 | 75 | 3 | 25 | 100 | 3.00% | 25.00% |
In order to standardize absorbances, the baseline was subtracted from each recorded post-incubation time per group. Values for replicate trials were then averaged. To analyze the difference between replicate trials the range between A and B was calculated and divided by the average to produce the deviation between replicate trials. Using the average absorbance bacteria per ml was calculated using the standard curve. Population generation time and the population doubling rate were then calculated for each group. The points used were the first point at the start of the exponential phase, and the last point at the end of the exponential phase. Linear regression was then performed to quantify the changes in both population generation time and the population doubling rate as a result of rockstar concentration.
The results from serial dilution and spectrometry show that absorbance and cells per ml have a positive correlation (figure 1). E.coli at a concentration of 1.13 million per ml exhibits an absorbance of .8 at 585nm. The following formula was calculated using linear regression to determine the correlation between E.coli per ml and absorbance at 585 nm.
x / 7.04*10^-10 = E.coli per ml
*with x being absorbance at 585nm