The Effects of Available Glucose Concentration on the Population Dynamics of Growing Escherichia coli Cultures. Abstract: Introduction:

Transcription

1 The Effects of Available Glucose Concentration on the Population Dynamics of Growing Escherichia coli Cultures 1 Shehzad H. Siddique and 2 Anne M. Vardo-Zalik, 1 Undergraduate student; 2 Penn State York Abstract: In this study we examined the relationship between the amount of available glucose per Escherichia coli cell and the growth rate of the bacterial population. This project was conducted as a pilot study to determine whether or not adding glucose to standard nutrient broth would increase the growth rates of microbial populations, such as E. coli, and to determine if there is an optimal glucose concentration for the growth of E. coli populations. We conducted four trials: standard nutrient broth with no added glucose, and nutrient broth with glucose added to a final concentration of either 1%, 0.5%, or 0.25%. The mass of the cell pellet and glucose concentration of the resulting growing cultures (for glucose trials) were analyzed at varying time intervals by centrifugation and subsequent titration of the supernatant with qualitative Benedict s solution. In all trials, the growth rates of the bacterial populations per time were found to exhibit characteristics analogous of third-order polynomials. In comparison to the control, glucose was seen to be initially inhibitory to the growth of bacterial populations. In the presence of glucose, however, bacterial populations of E. coli cells attained higher population densities, with their fastest rate of growth occurring at ± g glucose/ g E. coli. The growth curves were found to be limited by both the glucose concentration remaining, which diminished exponentially, and also by residual E. coli cells that have died. In this experiment we were able to show that, unlike non-limited bacterial populations, growing bacteria populations follow exponential growth initially, but this growth levels off typical of a third-order polynomial. Introduction: Glucose is one of the primary molecules which serve as energy sources for almost all organisms, including bacteria. However, one of the more common growth media used in microbiology labs, nutrient broth, does not contain glucose as the main carbon source for 7

2 bacteria (it contains protein). The addition of glucose to nutrient broth may increase the overall growth rates and biomass of bacteria over time. If so, this could be beneficial for lab purposes in that less time would be needed to grow cultures for experiments. Alternatively, glucose may be inhibitory to cell growth if the concentration is too high. Increased concentrations of glucose have been shown to limit bacterial growth by inhibiting proteinaceous enzymes; this strain on protein degradation may limit growth of bacterial populations in sugar solutions by reducing a cell s ability to breakdown and reincorporate proteinacious resources [1 & 2]. As of yet, no studies have published data on a specific concentration of glucose recommended for optimal growth of E. coli populations, however a 1% solution is referred to in some microbiology texts. This pilot research study set out to examine if adding glucose to nutrient broth increased the initial growth rates of Escherichia coli cells. This study analyzed the effects of glucose concentration on the population dynamics of E. coli (growth rate and the maximum biomass obtained) to determine if adding glucose to standard culturing media would be beneficial in practice. Methods: For each trial, Escherichia coli cells were cultured in test-tubes with 10mL of trialspecific liquid media, i.e. a combination of nutrient broth and the trial-specific glucose concentration. The trial-specific media was created by taking 10mL of nutrient broth and adding the appropriate amount of glucose to reach a final glucose concentration of 0%, 0.25%, 0.5%, or 1%. For each trial, fifteen tubes were inoculated, but not all were analyzed due to time constraints (15 were analyzed for the 1% trial, 11 for the 0.5% trial, 13 for the 0.25% trail, and 6 for the control). The # of cells in the inoculants were found by analyzing the stock solutions under a light-microscope with a haemocytometer; the inoculants for the 1%, 0.25%, and control trials were approximately 9.48x10 4 cells, whereas for the 0.5% trial the inoculant was 6.11x10 4 cells. All samples were analyzed between 20 and 80 hours post inoculation. Periodically, one random test-tube from each trial was centrifuged, and, for the glucose trials, the supernatant was analyzed for the concentration of glucose by titration with qualitative Benedict s solution. The concentration of glucose was found by dispensing the trial supernatant into a known quantity of Benedict s solution until the blue Benedict s solution turned clear. The amount of trial specific 8

3 supernatant needed was then compared to a standard curve created from known glucose concentrations, and the glucose concentration for the trial sample was determined. After centrifugation and removal of the supernatant, the remaining pellet of E. coli cells for all treatments was analyzed for its mass. A digital balance was used and the weight of the pellet, to the nearest g, was recorded after tarring the balance for the mass of a single testtube. The final masses were transformed into a plot of the number of E. coli cells vs. time by using a standardized wet-mass of E. coli cells: 9.5x10-13 grams/ E. coli cell [3]. In addition, the data collected was used to derive third-order polynomials to describe the population of E. coli cells vs. time and exponential functions to describe the concentration of glucose vs. time. It is important to note that the time-specific masses obtained for each trial consist of both living and dead cells; therefore, the plot of the number of E. coli cells vs. time for each trial shows both living and dead cells. No statistical analysis could be performed as only 1 replicate per time per treatment was performed in this pilot study. Therefore, all conclusions are subjective. Results: In all trials, population growth was found to exhibit characteristics analogous of thirdorder polynomials (Figure 1). The control trial was shown to have the highest initial growth, but the lowest maximum biomass. Glucose trials were found to have greater overall growth and maximum biomass than compared to the control trial. Due to the pilot-nature of this research project, no statistical analyses were able to be conducted to verify the results presented herein, therefore, the findings presented here are simply a preliminary look at the effects of glucose concentration on E. coli growth rates over time. Increased initial glucose concentration was shown to be positively correlated with increased maximum biomass (Figure 1). Initial exponential growth of Escherichia coli populations co-occurred with the initial exponential decrease of glucose in solution (Figure 2). In all glucose trials, growth rate was shown to peak sharply at ± g glucose/g E. coli cells. As expected, glucose concentration decreased exponentially in regard to time (Figure 2). For each trial, the stabilization of the glucose concentration occurs at nearly the identical time for the leveling off of its respective E. coli population (Figure 1). 9

4 Figure 1: The derived third-order polynomials for all four glucose trials are shown. Figure 2: The glucose molarity vs. time plots for all four glucose trials are shown. 10

5 Discussion: In the absence of glucose, growing Escherichia coli populations were found to exhibit higher initial growth rates, implying that glucose has an inhibitory effect on the early stages of growth (even at low glucose concentrations). Therefore, this preliminary project suggests that the addition of glucose to nutrient broth does not appear to be beneficial for microbial culturing practices if the intent is to increase initial growth rates. In the presence of glucose, growth rates were initially hampered, but were higher longterm with the growth rate peaking at a specific concentration of ± g glucose/ g E. coli cells (Figure 1). Overall, it appears that the bacteria grown in the 1% glucose solution attained highest growth rates and overall biomass. Glucose concentration decreased over time and reached a plateau around the same time period that the bacteria growth rates started to slow (Figures 1 & 2). A plausible explanation for this phenomenon is that the consumption of glucose by growing E. coli populations has caused glucose levels to drop to such a level that they can no longer support continued growth of the bacterial populations. Also, the accumulation of byproducts, such as acetic acid, and the decline in other food resources in the cultures can also contribute to this plateau [1]. What our preliminary results suggest is that glucose has an initial inhibitory effect on the growth rates of E. coli. The inhibitory effect suggested in this research project may be caused by the inhibition of catabolic enzymes involved in the breakdown of proteinaceous molecules, which would otherwise be incorporated for continued cell growth of E. coli cells. Previous research has supported the inhibitory effects of glucose on bacteria growth [1, 2, & 4]. For example, concentrated sugar solutions have not only been shown to reduce microbial growth when used as a food preservative, but they have also been applied to wounds to create an unfavorable environment for bacterial agents [4]. Our results imply that E. coli populations are able to offset the initial inhibitory effects of glucose and ultimately attain higher growth rates. Future research on this topic should first involve a replication of this study to determine whether or not the observations from this study hold, and whether these differences in growth rates are statistically significant. From this preliminary study, it appears that adding even a small amount of glucose to standard nutrient broth culturing media will not increase initial growth rates of an E. coli culture. 11

6 Acknowledgements: The authors would like to thank the Penn State York Science Department, as well as the Penn State York Honors Program for funding this research project. References: 1. Boyd, W.L., and H.C. Lichstein (1951). The Inhibitory Effect of Glucose on Certain Amino Acid Deaminases. University of Minnesota. Journal of Bacteriology, 62 (6): Kendall, A. I., and C.J. Farmer (1912). The Influence of the Presence of Glucose during Growth on the Enzymic Activities of Escherichia coli: Comparison of the Effect with that produced by Fermentation Acids. Journal of Biolchemistry, 36(7-9): Neidhardt, F. C., Ingraham, J. L., and Schaechter, M. (1990). Physiology of the bacterial cell. Sinauer Associates Inc. 4. Chirife, J., Herszage, L., Joseph, A., and E. S. Kohn (1983). In Vitro Study of Bacterial Growth Inhibition in Concentrated Sugar Solutions: Microbiological Basis for the Use of Sugar in Treating Infected Wounds. Journal of Antimicrobial Agents and Chemotherapy, 23 (5):