MICROBIAL DEGRADATION OF ...

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Achromobacter sp. (ATCC 21910) was obtained from the American Type Culture. Collection (ATTC) while Rhodococcus B30 and J10 strains were revived from ...
GENERAL PAPERS Water Session Organized by A.M. Ford (Pages 43-45 in Preprints of Extended Abstracts, Vol. 39 No. 1) Symposia Papers Presented Before the Division of Environmental Chemistry American Chemical Society Anaheim, CA March 21-25, 1999 MICROBIAL DEGRADATION OF METHYLHYDRAZINE CONTAMINATED NASA WASTEWATER A.U. Nwankwoala,* N.O. Egiebor,† C. Gilbert,† and K. Nyavor† Divison of Natural Sciences, Purchase College, State University of New York, Purchase, NY 10577 † Environmental Engineering Program, Tuskegee University, Tuskegee, AL 36088 *

Introduction Hydrazine (Hz) and its derivatives, monomethyl hydrazine (MMH) and 1,1-dimethyl hydrazine (UDMH) are widely used as rocket fuels, missile propellants and in the agricultural and pharmaceutical industries.1 These use have led to the inadvertent release of the chemicals in the environment and the accumulation of large volumes of industrial wastewater containing toxic levels of the hydrazine fuels. The carcinogenicity of the hydrazines to laboratory animals2 as well as their determined and potential hazards to humans has led to the concern for their fate in the environment. In addition, there is the task of finding appropriate cost-effective methods for the treatment and safe disposal of industrial wastes containing high levels of effluent hydrazine fuels. At the NASA-Kennedy Space Center in Florida, vent gaseous nitrogen (GN2), contaminated with hydrazine and methylhydrazine vapors, are scrubbed with citric acid solution to absorb the hydrazines (hydrazine and methylhydrazine) in scrubber liquor. While the cleansed GN2 is vented to the atmosphere, environmentally hazardous citric acid/hydrazine mixtures are being accumulated, posing decontamination challenges. Biological degradation of these hydrazine-containing wastes is probably the most cost-effective onsite-site treatment option. In this study, we investigated the microbial degradation of NASA’s scrubber water containing mixtures of methylhydrazine and citric acid solutions.

Materials and Methods Achromobacter sp. (ATCC 21910) was obtained from the American Type Culture Collection (ATTC) while Rhodococcus B30 and J10 strains were revived from previous isolates preserved in frozen agar. Achromobacter sp. was grown in ATCC Culture Medium 457 while the Rhodococcus sp. were grown in a modified Basal Salt Medium with glycerol as the major source of carbon. Microbial growth in the batch cultures was monitored by UV/Visible spectrophotometry, turbidimetry and total organic carbon measurements. Concentrated cells for biodegradation experiments were harvested from the cultures by differential centrifugation. The media composition and the detailed experimental procedure are reported elsewhere.3 Biodegradation of NASA wastewater was carried out in both batch cultures on an orbital shaker and on trickle-bed reactor columns. Details of the design and operation of the Trickle-Bed Reactor is provided in an upcoming publication.4 Samples were collected from the batch cultures and the feed reservoir on the trickle-bed set-up at regulated intervals and after appropriate treatments, analyzed by HPLC, TOC and UV/Vis. The UV/Vis method was used for the analysis of methylhydrazine at 455 nm after derivatization with p-dimethylaminobenzaldehyde. Upon completion of the degradation experiment, the samples were in dichloromethane (DCM) and analyzed by GC/MSD after concentration in a rotary evaporator and derivatization of the acidic fractions. Results and Discussion The HPLC analysis of the industrial NASA wastewater is shown in Figure 1. The wastewater in use for this work was accumulated when methylhydrazine was the predominant propellant in use at the NASA Kennedy Space Center and analysis of pure samples of citric acid and methyl hydrazine led to the assignment of the major bands as shown in Figure 1. In addition to the three major organic substances, the industrial wastewater contains traces of other species that are evident in the HPLC chromatogram.

Figure 1. HPLC chromatogram of methylhydrazine contaminated NASA wastewater. Figure 2 shows the variations in total organic carbon present in NASA wastewater during degradation in batch cultures and the Trickle-bed reactor by Rhodoccocus B30. In the batch cultures, an acclimation period of less than 2 days was followed by rapid degradation of the organic substances. Also, the consumption and mineralization of the organic carbon present in NASA wastewater was more rapid with both the B30 and J10 strains of the Rhodococcus sp than with the Achromobacter sp.

Figure 2. Changes in total organic carbon (TOC) concentration during the degradation of NASA scrubber water by Rhodococcus B30 in batch cultures and trickle-bed reactor columns. As expected, continuous passage of the wastewater through the trickle-bed reactor produced an enhanced degradation of the organic carbon by the three microbes. The kinetic plots showed an initial degradation reaction that proceeded at a very fast rate followed by a slower degradation process that leads to an almost limiting consumption. More than 50% of the organic carbon were degraded within the first 2 days and the limiting concentration (~ 85% degradation) for the biodegradation was achieved after

only about 4 days. It is important to note that control trickle-bed columns that had no immobilized microbes did not exhibit any significant changes in carbon concentration during continuous flow-through runs. This show that the depletion in the carbon contents observed in the experimental columns were indeed of microbial origin. Figures 3 show the degradation curves for methylhydrazine after inoculation by Rhodoccocus B30 in batch cultures and following continuous passage through the trickle-bed reactor column. Similar plots were obtained for the citric acid and the methyl hydrazine-citric acid reaction product present in the mixture. The Rhodococcus B30 was found to be most effective in the degradation of methylhydrazine in batch cultures among the three microbes used. About 40% of the methylhydrazine residues present in the wastewater matrix were degraded by this microbe within the first 15 days of microbial incubation. The Rhodococcus J10 exhibited similar behavior though degradation was at a slower rate than that observed for the B30 strain. All three microbes showed longer lag period for methylhydrazine degradation in comparison to citric acid degradation in the batch cultures. A postulated reaction product of methylhydrazine and citric acid, present in the wastewater, was also a target of rapid degradation by both strains of the Rhodococcus sp. The inclination of the microbes to preferentially utilize one major component of the NASA wastewater as a substrate makes mixed culture biodegradation of the industrial waste particularly viable.

Figure 3. Degradation of methylhydrazine present in NASA fuel wastewater by Rhodococcus B30 in batch cultures and trickle-bed reactor columns. Both Achromobacter sp. and Rhodococcus B30 were effective in the degradation of methylhydrazine along the trickle-bed columns. The degradation of methylhydrazine was also found to be very rapid during the first 4 days and gradually slows down to a limiting rate. By the end of the 12th day, the Achromobacter sp. and the Rhodococcus B30 had degraded 50% and 74% respectively, of the methylhydrazine, relative to the starting concentrations. Degradation of the wastewater by Rhodococcus J10 approximately mirrors the results obtained with the Rhodococcus B30 as plotted in Figure 3. It is believed that the degradation was essentially through the process of

mineralization since no metabolites were identified either by HPLC or GC-MSD techniques. Conclusion The results of this study show that NASA wastewater containing methylhydrazine propellant residues and citric acid as well as their reaction product are amenable to microbial degradation. Both the citric acid and the methylhydrazine residues present in the wastewater in addition to their reaction product were all depleted significantly by Achromobacter sp. and Rhodococcus B30 and J10. The Rhodococcus B30 was found to be most effective in degrading the methylhydrazine while the Rhodococcus J10 exhibited more versatility. The continuous passage of the wastewater through the trickle-bed column afforded higher levels of degradation of the methylhydrazine and the other organic constituents than inoculations in batch cultures. Acknowledgement The support of this work by a grant from the National Aeronautics and Space Administration (NASA Grant #NAG10-0176) and the supply of the industrial wastewater are gratefully acknowledged. References 1. Schmidt E.W. (1984) Hydrazine and its derivatives. John Wiley & Sons, New York. 2. IARC Monographs (1974) Evaluation of carcinogenic risk of chemicals to man, Vol. 4, International Agency for Research on Cancer, Lyon. P. 127. 3. Nwankwoala A.U., Egiebor N.O., Gilbert C. and K. Nyavor. Batch culture biodegradation of methylhydrazine contaiminated NASA wastewater. Biodegradation (In Press) 4. Nwankwoala A.U., Egiebor N.O. and K. Nyavor. Degradation of hydrazine contaminated NASA wastewater in fixed-film bioreactors. Appl. Microbiol. Biotechnol. (Manuscript submitted.)

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