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The Role of Microbial Communities in the Breakdown of Wastewater Pharmaceuticals

Lena Intro

Introduction

One of the most important scientific issues is human impact on the environment. Increasingly, pharmaceuticals are being detected in the environment because treatment plants and septic tanks are not always effective at removing them from wastewater (Kasprzyk-Hordern et al. 2009). The microbial communities in treatment facilities are diverse and difficult to monitor (Wagner et al. 2002). The purpose of the experiment described here is to address whether the diversity of bacterial species during water treatment affects the breakdown of pharmaceuticals. If not broken down during wastewater treatment, pharmaceuticals endanger wildlife. For example, some pharmaceuticals are endocrine disruptors, which can be especially harmful for the growth and reproduction of aquatic life. Endocrine disruptors appear to be responsible for reducing populations of some aquatic bird species because of thin-walled eggshells and kidney failure (Clouzot et al. 2008). Pharmaceuticals in the environment may leak into sources of our drinking water (Heberer et al. 2001). For example, pharmaceuticals have been found in Sebago Lake, the source of drinking water for Portland, Maine (Richardson 2009). Release of pharmaceuticals into the environment is not regulated in the Portland Wastewater District because it is not required in Maine (Jackson 2009). Improved wastewater management methods are needed to protect wildlife as well as sources of drinking water.

how pharmaceuticals enter the environment

Appendix A: This illustration depicts how pharmaceuticals enter the environment and are spread throughout the environment affecting many forms of wildlife


Background

Pharmaceuticals have short half-lives and degrade quickly, but may still contaminate the environment if constantly replaced (Rahman et al. 2009) (Appendix A). Pharmaceuticals have been detected in surface waters (Kasprzyk-Hordern et al. 2009), soils irrigated with wastewater (Kinney et al. 2006), biosolids (Ramires et al. 2007), groundwater (Barnes et al. 2008), and in wildlife. Examples of wildlife include fish in a river that had a large amount of sewage plant effluent (Ramires et al. 2007) and earthworms in farms irrigated with wastewater (Kinney et al. 2008).

The primary source of pharmaceuticals in the environment is wastewater (Kim et al. 2007, Kasprzyk-Hordern et al. 2009). Some pharmaceuticals are not completely metabolized by humans before being excreted (Barnes et al. 2008). Also, the disposal of pharmaceuticals by individuals may contribute to their presence in the environment. An interview found that 11.5% of people disposed of leftover pharmaceuticals in a sink or toilet (Bound et al. 2005).

Prokaryotic microorganisms are the main organisms removing pharmaceuticals during wastewater treatment (Wagner et al. 2002), and wastewater treatment is one of the most important technological processes utilizing bacteria (Juretschko et al. 2002). The activated sludge process (adding oxygen to wastewater) is the most efficient method for breaking down pharmaceuticals (Kasprzyk-Hordern et al. 2009). But even this process is not always effective, and bacteria can cause intermittent problems, such as bulking and foaming, which prevent the sludge from settling. Insufficient bacterial diversity in the treatment plant can cause low productivity (Wagner et al. 2002). In one experiment, 53 different bacteria groups were found in a treatment plant, all of which were previously known species (Juretschko et al. 2002). Under some conditions, the bacteria that break down specific pharmaceuticals may be out-competed by other species. More research is needed to determine why wastewater treatment plants are not always effective and to understand the role that microbial communities play.

The exemplar pharmaceutical used in this experiment is the compound chalcone. Its structure includes a three-carbon chain and an oxygen molecule joining two aromatic rings.

Chemical structure of Chalcone

Chemical structure of Chalcone


is a natural compound used as a pharmaceutical in some homeopathic medicines. Its uses include treatment of rheumatism, pain, ulcers, and cardiovascular diseases (Zdzislawa 2007). Preliminary data found that chalcone has a significant absorbance peak of about 316 nm (Appendix B).

Introduction of the Experiment

The purpose of this experiment is to examine the microbial communities of wastewater treatment facilities and how they break down pharmaceuticals to help understand why the treatment process may not always be successful.

This experiment has three parts. In Part 1, bacteria from a treatment plant and a septic tank were plated and compared to determine the microbial species diversity. The independent variable is the different treatment ecosystems, and the dependent variable is microbial diversity. Similar research was done by Juretschko et al. (2002), but it was suggested that it should be performed again under different conditions. In Part 2, several bacterial species were isolated and compared by how well they break down chalcone. Perhaps specific species are responsible for breaking down specific pharmaceutical compounds (Wagner et al. 2002). The independent variable is the species of bacteria, and the dependent variable is their efficiency in breaking down chalcone. In Part 3, the ability of the bacteria in the two different systems to remove a pharmaceutical will be compared. The independent variable is the type of ecosystem (the aerobic activated sludge of the treatment plant versus the anaerobic septic tank) (Jackson 2009), and the dependent variable is their ability to breakdown chalcone. These three parts will help determine whether bacterial diversity in a wastewater treatment plant affects the efficiency with which pharmaceuticals are broken down, and how and why this occurs.

Some variables kept constant throughout this experiment are the growing conditions of the bacteria, including nutrients, temperature, lighting, sample volume, bacterial number used in each assay (bacteria will be stored in frozen aliquots and thawed as used), and everything will be kept sterile (Jackson 2009). Also, the type and amount of the pharmaceutical used in the experiment will be kept constant. Some other constants will be where the samples are taken from and the time intervals at which the amount of pharmaceutical in the culture is measured (Kasprzyk-Hordern et al. 2009).

Based on previous experiments, it is hypothesized that the composition of bacterial species in a treatment facility will affect the efficiency of pharmaceutical removal. In Part 1, it is hypothesized that there is a significant diversity of bacteria in different treatment facilities because there are a variety of bacteria in treatment plants (Juretschko et al. 2002). In Part 2, it is hypothesized that certain bacterial species will be responsible for breaking down certain pharmaceutical chemicals because some individual bacteria have been found to breakdown certain compounds (Wagner et al. 2002). In Part 3, it is predicted that different treatment ecosystems will have different abilities to remove chalcone.

Materials and Methods

Appendix C: This graph shows the graph of a spectrophotometer with the visual light spectrum colored in.

Appendix C: This graph shows the graph of a spectrophotometer with the visual light spectrum colored in.


Initially, five samples were obtained from a home septic tank and three from PortlandWastewater Treatment Plant. The Portland Wastewater Treatment Plant uses the activated sludge process to add oxygen to the sludge (Kasprzyk-Hordern et al. 2009) and it serves 100,000 people (Jackson 2009). In the first step in this experiment, chalcone was dissolved in DMSO (dimethyl sulfoxide). A spectrophotometer (Shimadzu Biospec-mini) was then used to detect the removal of chalcone. Spectrophotometers measure the absorbance of light through the visual and ultraviolet range. Absorbance isdirectly proportional to the concentration. In preliminary experiments it was determined that the nutrient medium, DMSO, and the natural mixture of the treatment facilities have significantly different absorbance than chalcone and have only an absorbance of about 0.1 at chalcone's absorbance peak of about 316 nm. (Appendix C).

These spectrophometer absorbance graphs showing the absorbance of the isolated LB nutrients, DMSO, and chalcone at wavelengths varying from 190 nm to 500 nm. These graphs show that there is not significant absorbance (greater than about 0.1 O.D.316) at chalcone's absorbance peak of 316 nm.

These spectrophometer absorbance graphs showing the absorbance of the isolated LB nutrients, DMSO, and chalcone at wavelengths varying from 190 nm to 500 nm. These graphs show that there is not significant absorbance (greater than about 0.1 O.D.316) at chalcone's absorbance peak of 316 nm.


In Part 1 of this experiment, bacteria from the two different treatment facilities were grown on petri dishes in LB nutrient medium (10 grams NaCl, 10 grams bacto tryptone, 5 grams yeast extract) at room temperature for three days. The plates were initially grown with the original mixture at concentrations of 100%, 1%, 0.01%, and 0.0001% to determine a concentration where the bacteria colonies can easily observed and counted. Colonies were visually categorized by their shape, color, size, and texture. To learn about the different types of bacteria, the shape of the bacteria were examined under a microscope at 1000x and the gram stain performed to categorize the bacterial species (Jackson 2009) (Appendix D).

In Part 2 of this experiment, six different species each from the septic tank and the treatment plant samples were grown on plates. The bacteria were re-suspended in water with a one-twentieth dilution of LB nutrients and three L of 50 mg chalcone dissolved in 1 mL DMSO per 1 mL of distilled water. The number of the bacteria in the liquid was determined by diluting the bacteria as above, plating the bacteria on LB, and counting the number of colonies formed. This allowed the number of bacteria to be kept constant (5 x 106/assay). For each experiment, a negative control was included lacking chalcone. Samples were measured at 0, 2, 18, and 24 hours for four trials each. During incubation the bacteria remained suspended because the tubes were vibrated using an air shaker. At each time point, bacteria were removed by centrifugation so they did not affect the absorbance. Then the absorbance was measured at chalcone's absorbance peak of 316 nm. This part measured how efficiently specific species removed chalcone. Samples from the treatment plant and septic tank were each tested four times.

Appendix D: Gram stain procedure

Appendix D: Gram stain procedure


Appendix E: Part One: Gram stain; Examining bacteria under a microscope

Appendix E: Part One: Gram stain; Examining bacteria under a microscope


Appendix E: Part Two: Isolated bacteria species; bacteria species 6,7,11,12

Appendix E: Part Two: Isolated bacteria species; bacteria species 6,7,11,12


Appendix E: Part Three: Comparison of septic tank and treatment plant bacteria (Left septic tank, right treatment plant)

Appendix E: Part Three: Comparison of septic tank and treatment plant bacteria (Left septic tank, right treatment plant)


Appendix F: Results Part One

Appendix F: Results Part One


Appendix F: Results Part Two

Appendix F: Results Part Two


Appendix H: Results Part Three

Appendix H: Results Part Three


In Part 3 of the experiment, samples of 3 L (of 50 mg chalcone dissolved in 1 mL DMSO) per 1 mL were put into the natural ecosystems of the treatment plant and the septic tank. The number of bacteria was kept constant because colonies had been grown and counted previously and aliquots of bacteria frozen. There was also a control culture without chalcone. Between time points, a shaker machine was used to vibrate the samples. The absorbance peak of chalcone, 316 nm, was measured with a spectrophotometer after 0, 2, 18, and 24 hours with four trials each to determine if all the bacteria in the different systems can break down chalcone (Appendix E).

Results

The results of Part 1 of this experiment showed a diverse group of bacteria in the septic tank ecosystem and a comparably diverse yet different group of bacteria in the treatment plant ecosystem. Six species from each ecosystem were identified and studied in greater detail. These bacteria were determined to be different species by classification of their visual characteristics on both a macro and microscopic level and whether they were gram-positive or -negative (Appendix F).

In Part 2, 12 different bacteria species characterized in Part 1 were analyzed for their ability to break down chalcone. All the cultures with different bacteria broke down chalcone slightly, by about 0.40 O.D.316 at the 24-hour period. One of the bacteria species in particular, Bacteria 12—which was a red coccus gram-negative bacteria—broke down chalcone successfully in 18 hours (a very fast rate). Chalcone was broken down to an absorbance of 0.167 O.D.316, which is very similar to the absorbance of about 0.150 O.D.316 of the negative control (the absorbance of the nutrient medium at 316 nm, which was stable throughout the culture period). Bacteria 8 also broke down chalcone to about half of its original amount, about 0.721 O.D.316.  (Appendix G).

The results of Part 3 showed that the mix of bacteria from a wastewater treatment plant removed nearly all the chalcone (about .0363 O.D.316), but the mix of bacteria from a septic tank did not reduce chalcone in a 24-hour time period. A statistical T-test was used on the values in Part 3 to compare the 0-hour time point to the final 24-hour time point to determine whether the values were significantly different. P-values less than 0.05 are considered statistically significant, and therefore the numbers at the 0-hour time point and the final 24-hour time point are different. For the treatment plant, the P-value was 0.000042129, supporting that the bacteria could break down chalcone. The P-value for the septic tank was 0.76839, supporting that chalcone was not broken down. The final time points of these two ecosystems were also compared with a T-test, and the resulting P-value of 0.00030529 supported that the two ecosystems gave different results. Bacteria 12 in Part 2, which successfully broke down chalcone, was only present in the treatment plant sample, but colonies with its distinctive red color were not observed on petri plates from the septic tank sample. This suggests that individual bacteria are responsible for the successful break down of chalcone, and if the appropriate species are not present, then chalcone cannot be broken down (Appendix H).

The results of Part 3 showed the septic tank ecosystem did not break down chalcone, but the six isolated bacteria species from the septic tank in Part 2 appeared to have some ability to break down chalcone (to about 0.40 O.D.316 by 24 hours). Perhaps this is because the isolated bacteria in Part 2 were exposed to different nutrients (LB nutrients and natural nutrients in Part 3). These different conditions may have favored different bacteria, or perhaps competition between bacteria species could have led to under-representation of the bacteria that broke down chalcone some. Given the small sample size, it is interesting to wonder whether, if more bacteria species were isolated, perhaps some could be isolated that would not break down chalcone at all.

Overall, the results of my experiment were successful, and many factors were controlled for. Some factors of my experiment that could be improved include trying to grow a wider range of bacteria. It would be best if the bacteria could be grown in their natural environment (e.g., anaerobic for the septic tank bacteria). Bacteria survive at different temperatures and with different nutrients, and only six species of bacteria were selected, so the bacteria used were only a small selected portion of all the species in a treatment facility. Other bacteria in both systems may also break down chalcone. Also, the removal of chalcone was measured, but what happened to the chalcone was not determined in this experiment.

Conclusion

The hypothesis was supported by the results. In Part 1, as hypothesized, a number of different bacteria species could be categorized in both the septic tank and the treatment plant. In Part 2 it was hypothesized that specific species would break down chalcone. All of the species broke down chalcone to some extent in 24 hours, but two bacteria broke down chalcone more efficiently. In Part 3, the hypothesis that the two different treatment facilities would have different abilities to breakdown chalcone was supported by the results, in which the treatment plant could break down chalcone, but the bacteria in the septic tank sample could not. Thus, the environment influences how well pharmaceuticals are broken down.

It is concluded from these results that there are a diversity of bacteria in treatment systems, and low efficiency in wastewater treatment can occur if the species that break down chalcone efficiently are under-represented because of environmental conditions or competition.  

Opportunities for further research include detecting pharmaceuticals in the environment to learn what areas and water supplies are polluted with pharmaceuticals. More information is also needed about the effects of some pharmaceuticals on wildlife. Perhaps more could be learned about how competition and growth conditions affect the diversity of bacteria by performing an experiment in which a sample is disturbed with an antibiotic that removes certain bacteria species, while the species of bacteria are examined over time. Another interesting topic would be to test which pharmaceuticals are most common in the environment, and to learn more about why these pharmaceuticals are not effectively broken down during wastewater treatment.

It is important to remove pharmaceuticals during wastewater treatment so they are not released into the environment. Since it is impossible to continuously test for the presence of every pharmaceutical, the results of this experiment suggest removal of pharmaceuticals might be best assured by monitoring and regulating the composition of the microbial communities in treatment plant ecosystems. Since it is impractical to monitor private septic systems, which are common where I live, it is very important to educate students and families that it is inappropriate to dispose of unused medicines down the drain and into their septic systems.

Acknowledgements

I would like to thank Laurel Jackson, an environmental scientist, and the Portland Water District's research lab for their help with my background knowledge and for supplying me with samples. I would also like to acknowledge the microbiology laboratory of the University of Southern Maine for use of their facilities, and Caryn Prudente, a chemistry professor at the University of Southern Maine, for her advice.

Bibliography

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