Chemistry Research

The research was completed at St. Henry High School from May 1,1993 to November 20,1994. Lisa Schlater was a St. Henry's graduate in 1995 and is currently attending Heidelberg College as a member of the Graduating Class of 1999 working towards a degree in Chemistry as a pre-med student. She has completed chemical research on several projects, namely "Refining Used Motor Oil by Removal of Hazardous Metal Contaminants" and "An Alternative Synthesis for Epothilone Precursors: A Recently Discovered Anti-Cancer Drug", under Dr. Esterline's tutelage.

ABSTRACT

The process of finding microorganisms that digest harmful contaminates found in our environment, better known as bioremediation, and utilizing them to clean-up specific contamination sites shows a great deal of promise. The objective of this project was to locate a microorganism native to the Ohio-Indiana area that is able to degrade the herbicide atrazine. Methods employed include incubation of creek-water samples during high atrazine load (spring time) on tricas soy agar (TSA) plates, ELISA immunoassay to determine atrazine levels, some HPLC analysis for attempted degradation-product identification, and various microbe identification tests. A microorganism was located and shown to be capable of degrading atrazine.

INTRODUCTION

Herbicides are increasingly being used to enhance crop production by controlling competing vegetation. The herbicide atrazine, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (Figure 1), is used throughout the world. Between 1980 and 1990 the United States used approximately 800 million pounds of atrazine.[1] The triazine ring found in atrazine is well known for being resistant to degradation under aerobic condition.[2] The extensive use of this herbicide along with the triazine's resistance to degrade greatly increases the chance for long-term environmental contamination; therefore, development of a safe and economical clean-up technique for point source atrazine spills is desirable. Biotechnology may hold the key to finding a safe solution for the clean-up of spills containing high concentrations of atrazine.

FIGURE 1: Structures of Atrazine and its Metabolites

BIOREMEDIATION

Bioremediation is an environmental clean-up technique that is currently being investigated for use on a wide variety of chemicals. Bioremediation is the use of naturally occurring microorganisms to enhance biodegradation, or normal biological breakdown. The results from current tests indicate that bioremediation is a safe, viable remedy for the detoxification of environmentally hazardous chemicals.[3] There are three primary approaches to bioremediation: biostimulation, bioaugmentation, and genetically-engineered microorganisms (GEM's).[4]

The most studied of the three approaches is biostimulation. Biostimulation involves the addition of nutrients that are deficient but required for biodegradation of the contaminate and is comparable to fertilizing a lawn. The addition of nutrients causes an increase of microbial populations, thereby, increasing the number of indigenous microorganisms capable of degrading the contaminate. This technique has been tested with success on 110 miles of shoreline contaminated with oil during the 1989 Exxon Valdez oil spill.[4]

Another method is bioaugmentation. Bioaugmentation is the addition of nutrients and microorganisms to a contaminated environment to increase the rate of biodegradation.[5] Normally the microorganisms applied are a mixture of specially selected microorganisms that have been chosen for their ability to ingest the contaminate. If a seed culture can stimulate the rapid onset of biodegradation, bioaugmentation would have a significant advantage over biostimulation because biostimulation relies on indigenous microbes that may need time to adapt to the new conditions of their environment. Seed cultures may be most appropriate for situations in which native organisms are either present as slow growers, or the native organisms are unable to degrade the contaminate.

The third and most controversial approach is GEM's, altering the genetic code of microbes in an attempt to stimulate an appetite for environmental contaminants. GEM's are currently being tested in laboratories. Many individuals, including EPA officials, believe that scientists are far from realizing the potential of naturally- occurring microorganisms to degrade contaminates. Thus, the increased problems associated with GEM's, such as higher cost and opportunity for disasterous results, render them unnecessary at this time.[4]

Bioremediation has been recognized as one of the more cost-effective and environmentally desirable clean-up alternatives. What makes bioremediation so desirable is that it's a permanent solution; it destroys the contaminant. Bioremediation focuses on detoxification rather than waste translocation.[5]

ATRAZINE

In Basel, Switzerland, in the 1950's J.R. Geigy Ltd. discovered the herbicidal properties of the s-triazines.[6] Atrazine is an s-triazine herbicide, which means the triazine ring is symmetrical. Atrazine has been found in many of the Midwestern United States in ground and surface waters making human exposure a concern.[7] Most of this contamination is due to runoff from fields in agricultural areas.

In terms of human exposure, contamination of ground water and soil from spills and negligence may be more significant than contamination from agricultural runoff.[8] Thousands of pounds of atrazine are stored at mixing and loading facilities in often less than ideal conditions. Most instances of herbicide contamination are the direct result of carelessness. A small amount of atrazine can contaminate a large aquifer. Twenty pounds of atrazine evenly dispersed throughout an aquifer is sufficient to contaminate 10 million liters (2.6 million gallons) of water at a level of 100 ppb (parts per billion).[8] This level is significantly above the accepted maximum contamination level in drinking water of 3 ppb.[9]

Atrazine is a selective herbicide used for control of grassy and broadleaf weeds in corn, sorghum, rangeland, and sugarcane crops. Atrazine is a restricted-use herbicide, which means applicators must have certification to purchase and use atrazine. This is due to ground water concerns and its toxicity to aquatic invertebrates.[10] Laboratory animals exposed to high concentrations of atrazine for long periods of time have shown signs of liver, kidney, lung, or cardiovascular damage.[11]

ATRAZINE DEGRADATION

The three main degradation pathways of atrazine that have been previously studied are biological dealkylation, chemical hydrolysis, and biological hydrolysis (Figure1). Complete degradation of atrazine requires many different reactions. No organism has been found to contain all the needed enzymes to completely degrade atrazine into harmless byproducts. Complete degradation so far has required communities of more than one microorganism.[12]

Biological dealkylation is the most extensively studied of the three pathways (Figure 2). The atrazine is first degraded through oxidative N-dealkylation. This process has been found to form three different metabolites: deisopropylatrazine (CEAT), deethylatrazine (CIAT), and desethyldesisopropylatrazine (CAAT). In order to simplify discussion of the atrazine metabolites, the names have been abbreviated based on the substituents on the triazine ring (Table 1). Each metabolite takes a slightly different pathway to a common intermediate product, OOOT.[12]

Table 1: Abbreviations for metabolites based on the substituents on the triazine ring.

Abbreviation	Substituent
A	amino
C	chloro
E	ethylamine
I	isopropylamino
O	hydroxy
T	triazine ring

Deisopropylatrazine is first transformed into EOAT and then to EOOT before it reaches OOOT. Deethylatrazine is transformed to IOAT and then to IOOT before it reaches OOOT. Before forming OOOT desethyldesisopropylatrazine is first changed to COAT and then to COOT. These intermediate reactions before reaching OOOT have been tentatively identified as deamination reactions (Figure 2).[12] The metabolite OOOT is then further degraded to biuret and then to urea. The urea is then broken down into carbon dioxide and ammonia.

FIGURE 2: Atrazine Metabolic Pathways in Microorganisms

The second pathway studied is chemical hydrolysis. Chemical hydrolysis of atrazine into hydroxyatrazine is the principle pathway of detoxification in soil and is conducive to ring cleavage and total breakdown by microorganisms.[13] First, dechlorination takes place. Once the chlorine is removed, a hydroxyl group (OH) takes its place. Reports have shown that this process is catalyzed by an organic catalyst.[1] After hydroxyatrazine is formed, microbial degradation takes over and produces a metabolite. The metabolite is then further degraded by microorganisms. After the process is complete, carbon dioxide and ammonia are left as waste products. The third pathway studied is biological hydrolysis. It was previously thought that hydrolysis could only occur chemically. In 1993 a study conducted by the Department of Biochemistry and Institute for Advanced Studies in Biological Process Technology and the Department of Soil Science at the University of Minnesota proved otherwise.[1] The study shows that the dechlorination of atrazine can not only be catalyzed by an organic catalyst but can also be mediated by bacterial enzymes. After the chlorine is removed an OH group is added forming hydroxyatrazine. Once hydroxyatrazine is formed microbial degradation continues until carbon dioxide and ammonia are left.

EXPERIMENTAL

The objective of this experiment was to locate a microorganism native to midwestern Ohio that can degrade atrazine. The first step towards completing the objective was to find a microorganism capable of tolerating high concentrations of atrazine. Water samples were obtained from a local creek. The creek runs through an agricultural area where atrazine and other agricultural chemicals were expected to have an effect on the indigenous microbes.

Experiment 1
The samples were placed on tricas soy agar (TSA) plates and incubated at room temperature for 24 hours. Mixtures of the resulting bacterial colonies were then streaked onto nutrient plates containing various concentrations of atrazine.

To determine whether the microbes were degrading the atrazine, 25 ml of minimal salts containing 12 g/L KH2PO4, 12 g/L K2HPO4, 4 g/L NH4Cl, 10 mg/L FeSO4 - 7H2O, 100 mg/L MgSO4 - 7H2O, were added to 11 numbered test tubes. Inoculant, atrazine, and glucose were added to certain test tubes as shown in Table 2.

Table 2: Contents of Test Tubes 1 Through 11

Test Tubes	Atrazine Solution (in mg/mL)	Glucose (in g)	Inoculant Added?
1	0.16	0	Yes
2	0.12	0	Yes
3	0.08	0	Yes
4	0.04	0	Yes
5	0.16	1.0	Yes
6	0.12	1.0	Yes
7	0.08	1.0	Yes
8	0.04	1.0	Yes
9	0	0	Yes
10	0.16	0	No
11	0.16	1.0	No

Glucose was added to test tubes 5 through 8 to determine if the microbes could degrade the atrazine but required additional nutrients for survival.

Initial samples from each test tube were taken and fixed to a microscope slide. After verification that microbes were present, the slides were saved for comparison with the final slides. The test tubes were then incubated at room temperature for 168 hours (7 days). A smear was taken, fixed to the slide, and compared to the initial slide. Incubation continued for one month. To determine the exact level of atrazine remaining in the test tubes, an immunoassay (a competitive ELISA) was performed.

The experiment was repeated with second generation microbes to confirm the initial results. The test tubes were numbered from 12 to 15 and filled with 25 mL of minimal salts. The same concentrations of atrazine were added as in test tubes 1 through 4 (Table 2). The experiment was then conducted in the same manner as the first.

A gram stain and an identification of shape were performed to identify the microbes. A smear of the microbes was place on a slide. The slide was air dried and flame fixed. A gram stain was performed by flooding the slide with crystal violet stain for one minute followed by a rinse with water. The slide was then flooded with potassium iodine for one minute and then rinsed with a 95% alcohol solution. The slide was flooded with a counter stain, saffarin, for one minute followed by a final rinse with water. The slide was microscopically examined to determine the results of the gram stain and also to identify the shape of the microbes.

Results

The initial water samples produced a large variety of colonies. After a cross section of the colonies were streaked onto nutrient plates containing atrazine, only two types of colonies remained.

As expected, the control (Test tube 9) showed no growth due to the absence of any food source. The second slide showed only dead cell mass. Microbial growth did occur in test tubes 1 through 8. The results obtained from a second run confirmed these results. The levels of atrazine remaining after one month in test tubes 1-4 and 10 (as labelled in Table 2) were determined by an immunoassay (Table 3).

Table 3: Immunoassay Results

Test Tube # (see Table 2)	Original Concentration (in ppb)	Final Concentration (in ppb)
10	160,000	28.4
1	160,000	15.9
2	120,000	22.5
3	80,000	13.5
4	60,000	11.0

The two remaining strains had different shapes and gram staining results. One strain was gram positive and coccus in shape; the other was gram negative and rod-shaped.

Between March 1 and May 1 the gram-positive coccus culture died. Therefore a second experiment was completed to determine if the gram-negative rod bacterium could degrade atrazine without the aid of the gram-positive microbe.

Experiment 2

Eight test tubes were filled with minimal salts. Two of the test tubes received 0.4 mL of the atrazine solution and two different test tubes were inoculated without atrazine to be used as controls. The four remaining test tubes were inoculated with the microbe and 0.4 mL of the atrazine solution. One gram of glucose was also added to two of the four remaining test tubes. The test tubes were incubated at room temperature and after one month an immunoassay for detection of atrazine was performed.

The immunoassay results did not show a significant difference between the amount of atrazine in samples from the control test tubes compared with samples from the experimental test tubes. Additionally, the presence or absence of glucose did not effect atrazine degradation. Since the gram-negative rod did not show any significant atrazine-degradative ability without the gram-positive coccus being present, it most likely degrades a byproduct of atrazine, not atrazine itself.

Experiment 3

A third experiment was completed in an attempt to find a single microorganism that can degrade atrazine. For the third set of experiments, a fresh water sample was taken from the same area where the initial sample was collected. The water sample was placed on TSA plates and incubated under previously described conditions. The resulting microbe colonies were then streaked on to nutrient plates containing atrazine (0.16 mg/ml). After an incubation period of three days, a single microorganism remained on the atrazine-laden plate. The resulting microorganism was inoculated into test tubes containing minimal salts and atrazine. After 30 days an immunoassay screening was performed on samples from both the control and the inoculated test tubes. Results from the immunoassay showed approximately 5% less atrazine in the inoculated test tube.

Various identification tests were conducted. A gram stain and identification of shape was performed. An oxidase and catalase test was then performed. The microbe was also inoculated into various media: glucose, acetate, and gelatin. The microbe was also tested for fluorescent pigments under a 254 nm ultraviolet light and for fermentation by inoculation into dextrose. Various flagella stains were also conducted.

Identificatiion of the two most common atrazine metabolites, namely CEAT and CIAT, was attempted employing High-Pressure Liquid Chromatography (HPLC) without success.

DISCUSSION/CONCLUSIONS

A microorganism exposed to very high concentrations of atrazine was found to degrade atrazine by approximately 5%. The results from the comparison between the first test to determine the degradation abilities was a good indication that the microbes were utilizing the atrazine as a food source. The increase in the number of microbes on the slide indicated that the microbes are able to survive on atrazine only. The control slides showed only dead cell mass which indicates that the microbes are not able to live off of salts alone and that the microbes must be using the atrazine as a food source. The results of the slides from the second generation of microbes, experiment 3, confirmed the results of the first test. Comparing the control (test tube #11) and the test containing high levels of atrazine (test tube #1), the atrazine level decreased by 12.5 ppb. These results, although not statistically significant, help to confirm the conclusion that the microbes are degrading some of the atrazine.

After the loss of the gram positive coccus the gram negative rod was tested to see if it alone could degrade atrazine (experiment 2). The result of the immunoassay screening confirmed that the rod could not degrade the atrazine and that the coccus was needed.

The gram positive coccus was not recovered but a different gram positive coccus was found that could grow on medium containing high concentrations of atrazine (experiment 3). After further study, the immunoassay confirmed that the microbe was able to degrade the atrazine. The HPLC results after 15 days also confirmed the fact that the atrazine was being degraded. A pathway of degradation was not able to be determined because the metabolites tested for were not present. The degradation was approximately 5% but when compared to the half life of atrazine in water, which is four years, this rate of degradation is very significant.

The atrazine-resistant microorganisms isolated in this study have not yet been positively identified. With further testing and development in this area of study, the use of microorganisms to clean-up atrazine spills will become possible. More research is needed to identify not only the pathway of degradation but also the potentially-harmful by-products that result. Because bioremediation is an excellent solution for clean-up of hazardous chemical spills, further exploration is vital.

ACKNOWLEDGEMENTS

I would like to thank the Water Quality Laboratory at Heidelberg College, specifically Ellen Ewing, for analysis of the competitive ELISA and the HPLC tests as well as their expertise in interpreting the results. I'd also like to acknowledge Robert Hiskey (Biology Professor at the Celina branch campus of Wright State University) for graciously providing the needed equipment, Dr. Daniel T. Esterline (Chemistry Professor at Heidelberg College) for editing my written research paper and preparing it for the Internet, as well as my St. Henry high school science teachers, namely Randy Hoying and Bill Steinbrunner, for their interpretation/explanation of the subject matter.

BIBLIOGRAPHY

1. Allan, Deborah L., Raphu T., Mandelbaum, and Lawrence P. Wackett, "Rapid Hydrolysis of Atrazine to Hydroxyatrazine by Soil Bacteria." Environmental Science & Technology, 27, (9), 1943-1946 (1993)
2. Jessee, J.A., R.E. Benoit, A.C. Hendricks., G.C. Allen, and J.L. Neal. "Anaerobic Degradation of Cyaburic Acid, Cysteine, and Atrazine by a Faculatative Anaerobic Bacterium." Applied and Environmental Microbiology, Jan. 1983: 97-102
3. Sims, Michael. Bioremediation Nature's Cleanup Tool. The Texas Land Commission, 1991
4. Bioremediation of Marine Oil Spills, Congress of the United States Office of Technology Assessment, Congressional Board of the 102d Congress, pg 1-29
5. Dzantor, E. Kudjo and Allan S. Felsot. "Combination of Landfarming and Biostimulation as a Waste Remediation Practice."
6. Mirgain, G.A. Green and H. Monteil. "Degradation of Atrazine in Laboratory Microcosms Isolation and Identification of the Biodegrading Bacteria." Environmental Toxicology and Chemistry, 1993, Vol.12, p. 1627-1634
7. Kalkhoff, Stephen J. and Dana W. Kolpin. "Atrazine Degradation in a Small Stream in Iowa." Environmental Science & Technology, Vol. 27, No. 1, 1993: 134-139
8. Bode, Loren E. "Agricultural Chemical Application Practices to Reduce Environmental Contamination." American Journal of Industrial Medicine, 1990: 485-489
9. Nair, Dhlleepan R. and Jerald L. Schnoor. "Effect of Two Electron Acceptors on Atrazine Mineralization Rates in Soil." Environmental Science & Technology, 1992, Vol. 26, p. 2298-2300
10. Technical Bulletin - AATREX HERBICIDE, Geigy Agricultural Chemicals, Division of CIBA-GEIGY Corporation, Ardsley, NewYork
11. "Atrazine-Eating Microbes Found." State Line Farmer, 14 December 1993 : B1
12. Cook, Alasdair M. "Biodegration of s-triazine xenobiotics." FEMS Microbiology Reviews. 1987, 46:93-116
13. Goswami, Kishore P. and Richard E. Green. "Microbial Degradation of the Herbicide Atrazine and its 2-Hydroxy Analog in Submerged Soils." Environmental Science & Technology, May 1971, Vol 5, p. 426-429

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Last modified: July 19, 1999. JG.