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Student involvement in bioremediation of organic laboratory waste.

Student involvement in disposal of waste generated by undergraduate laboratory exercises in organic chemistry generally ends by placing material no longer needed in a provided container, which is disposed of by the sponsoring chemistry department. This study introduces a bioremediation method (Bouer & Zehnder 1993) for organic waste that can be successfully accomplished as an undergraduate research project. This approach simultaneously promotes an appreciation for multidisciplinary studies and elevates the environmental consciousness of students.


Naturally occurring bacteria were collected from a variety of sources including soil, wastewater sludge, septic effluent and a grease trap. The bacteria were collected and grown in standard media. The bacteria were then isolated by centrifugation, washed with buffer, recentrifuged, and then suspended in a mineral salt solution purchased from Sigma Chemical Co. These bacteria were grown in the mineral salt solution with ethyl butyrate as their sole carbon source. Ethyl butyrate was added to a total concentration of 1% to the inoculated medium. The flasks were capped with cheese cloth (for aeration) and placed in an incubator/shaker for 48 hour periods. At the end of each period, the absorbance at 600 nm was monitored on every flask to observe bacterial growth.

To test for the continued presence of ester in the bacterial cultures grown with ethyl butyrate as the sole carbon source, a 1.0 mL aliquot of culture was analyzed for ester. To the 1.0 mL aliquot, 0.4 mL of [H.sub.2]NOH*HCl in C[H.sub.3]OH was added. 2.0 M KOH was added dropwise until the solution was basic. The solution was then passed over a flame until it began to boil. It was immediately removed from the heat and allowed to cool to room temperature. 2.0 M HCl was added dropwise until the pH was around three, then one drop of 10% Fe[Cl.sub.3] was added. A magenta color is indicative of a positive test for ethyl butyrate, and a yellow color is a negative result (Cheronis & Entrikin 1963). A standard ethyl butyrate solution was made. Serial dilutions were then made, and a Beer's Law Plot was constructed to try to determine the concentration of ester remaining over time in the bacterial cultures.

Erlenmeyer flasks were prepared with 250 mL of the mineral salts solution with 1% ethyl butyrate as the sole carbon source. Approximately 2.5 mL of the bacteria from overnight cultures were suspended in phosphate buffer and added to one set of the flasks. The control set of flasks contained the mineral salt solution and had no bacteria added. The flasks were incubated and bacterial growth was monitored for 20 days at 600 nm. Every two days an aliquot was removed for extraction of the ester, and the flasks were returned to the incubator.

The extraction of the ester from the media was carried out by removing 1.0 mL from each flask. 2.0 mL of diethyl ether was added to the 1.0 mL aliquot. This mixture was shaken, vented and the broth layer was removed. The ether layer was then filter sterilized using a 0.22[micro]m filter to remove any remaining bacteria, diluted to a final volume of 5.0 ml with diethyl ether, and stored at -20[degrees]C until further analysis by gas chromatography (GC) could be carried out.

Ethyl butyrate standards of 500 ppm, 250 ppm and 100 ppm were prepared and analyzed by GC using a Varian Star 8200 CX autosampler, which was attached to the Varian Star 3600 CX GC/MS. Retention times for ethyl butyrate were determined from these standards. The aliquots that had been removed every two days, extracted and stored in the freezer were also analyzed by GC. The concentration of the samples were determined.


The use of visible spectroscopy to monitor the growth of bacteria at 600 nm demonstrated that the bacteria from the wastewater sludge did in fact grow using ethyl butyrate as their sole carbon source. Preliminary characterization using standard microbiological techniques of these bacteria indicate it is a Bacillus that replicates in the presence of ethyl butyrate as its sole carbon source. Bacteria from other sources did not grow significantly.

The classic qualitative test for ester using the ferric hydroxamate method demonstrated that the presence of ester in the bacterial cultures could be monitored over time. The ability to generate Beer's Law Plot also indicated that this qualitative test could possibly become a quantitative test; however, the amount of culture needed and the time to perform the ferric hydroxamate test were cumbersome.

The GC analysis showed that ester could in fact be detected; however, the expected, consistent decrease in ethyl butyrate concentration as the cultures grew was not observed. The MS showed an increase in the total mass over time which suggestive of microbial growth. These results were compatible with those from the visible spectroscopy for bacterial growth when ethyl butyrate was used as the sole carbon source.

The results of the mass spectrometer and visible spectrophotometer analysis showed the bacteria did grow in the flasks with ethyl butyrate as the only carbon source. Since ethyl butyrate was the only carbon source, it is deduced that these bacteria must be metabolizing the ethyl butyrate. The ferric hydroxamate test indicates it will be possible to not only detect the ethyl butyrate but may also provide another quantitative assessment it with further work. It is believed the extraction technique for ethyl butyrate from the growing cultures will need to be refined to reliably measure the concentration of ethyl butyrate by GC analysis. Future GC analysis may be performed immediately after the extraction to determine if storing in the freezer affected the sample. Currently, it is not known if ethyl butyrate is being metabolized to C[O.sub.2] and [H.sub.2]O, or to other products. GC/MS analyses will also be used to determined what metabolites are being produced from the breakdown of ethyl butyrate by bacteria.


The Robert A. Welch grant to the Stephen F. Austin State University Chemistry Department supported this work. Dr. Steve Wagner of the SFASU Biology Department provided helpful insight and supplies throughout this project.


Bouer, E. J. & A. J. Zehnder. 1993. Bioremediation of Organic Compounds--Putting Microbial Metabolism to Work. Trends in Biotechnology, (8) 360-367.

Cheronis, N. D. & J. B. Entrikin. 1963. Identification of Organic Compound. John Wiley & Sons Inc., New York, 140-141 pp.

Andrew Awtry, Amber Stiles, James M. Garrett and Michele R. Harris

Department of Chemistry, Stephen F. Austin State University

Nacogdoches, Texas 75962

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Title Annotation:GENERAL NOTES
Author:Awtry, Andrew; Stiles, Amber; Garrett, James M.; Harris, Michele R.
Publication:The Texas Journal of Science
Geographic Code:1USA
Date:Aug 1, 1999
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