Optimization of an Aerobic Polishing Stage To Complete the

The addition of an aerobic stage to remove excess external carbon after the anaerobic ... Zhao , Chuan-Shu He , Hou-Yun Yang , Li Gong , Yang Mu , Han...
0 downloads 0 Views 231KB Size
Download PDF
Environ. Sci. Technol. 1996, 30, 2021-2026

Optimization of an Aerobic Polishing Stage To Complete the Anaerobic Treatment of Munitions-Contaminated Soils DEBORAH J. ROBERTS,* FARRUKH AHMAD,† AND SUHASINI PENDHARKAR‡ Department of Civil and Environmental Engineering, University of Houston, Houston, Texas 77204-4791

The addition of an external carbon source to allow the creation of anaerobic conditions for the remediation of soils contaminated with nitroaromatic compounds has been successfully applied to soils contaminated with Dinoseb (2-sec-butyl-3,4-dinitro-ocresol), an herbicide, and 2,4,6-trinitrotoluene (TNT), an explosive. The addition of an aerobic stage to remove excess external carbon after the anaerobic stage produces a treated soil with a lower oxygen demand than the soil, which is presently left after the anaerobic stage. The use of acetate, soluble starch, glucose, and insoluble starch as sources of external carbon for the creation and maintenance of anaerobic conditions was examined. The addition of glucose to statically incubated soil reactors allowed for the fastest reduction in redox potential and produced cultures with the lowest redox potentials (-400 mV). The amount of glucose added was optimized resulting in the use of 0.25% (w/v) glucose to treat a sandy soil contaminated with 12 000 mg of TNT, 3000 mg of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), and 30 mg of HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine)/kg of soil. In these treatments, the anaerobic stage was complete within 14 days, and an additional 7-day aerobic stage resulted in TOC concentrations of 30 mg/L remaining in the aqueous phase.

Introduction Compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7tetranitro-1,3,5,7-tetraazocine (HMX) are found as soil contaminants at many facilities where munitions are produced and disposed of (1-3). These compounds are persistent in aerobic soils and leach into groundwater from contaminated soils (2, 4, 5). TNT or its reductive inter* Corresponding author telephone: 713-743-4281; fax: 713-7434260, e-mail address: [email protected]. † Present address: 800 Babcock No. S4, San Antonio, TX 78201. ‡ Present address: 1586 West Maggio Way, Apartment 9-2082, Chandler, Arizona 85224.

S0013-936X(95)00814-5 CCC: $12.00

 1996 American Chemical Society

mediates are mutagenic (6, 7) and toxic to plants, (8), fish (9, 10), larvae (7), algae (7, 9), microorganisms (7, 11, 12), and fungi (13-15). The remediation of soils contaminated with these compounds is important for the protection of critical groundwater resources as well as for demilitarization of Department of Defense (DOD) facilities. Incineration is presently the most often used method for treatment of these soils (16), but is expensive and not well accepted by the public. The development of an effective, environmentally sound, and economical treatment method to remediate munitions-contaminated soils is important to allow the cleanup of these numerous sites. The biological degradation of TNT has been investigated by many research groups and has been the subject of several reviews (3, 17-20). Aerobic treatment processes generally result in the conversion of TNT to aminodinitrotoluenes, diaminonitrotoluenes, or tetranitroazoxytoluenes. These compounds are more persistent and in some cases more toxic than TNT and may represent slow release reservoirs for the release of nitroaromatic compounds. TNT is also metabolized under anaerobic conditions through reductive mechanisms, but due to the absence of O2 and the rapid reduction to diaminonitrotoluenes or triaminotoluene, relatively low concentrations of the hydroxylamine intermediates are present, and the formation of tetranitroazoxytoluenes is very low if it occurs at all. The anaerobic degradation of TNT to TAT (21), toluene (22-25), or further through p-cresol (26, 27) have been reported. A soil remediation procedure developed for the anaerobic biological remediation of soils contaminated with the nitroaromatic herbicide Dinoseb (28-30) has been applied successfully to the treatment of soils contaminated with TNT and RDX (31-33). In this procedure, an external easily degradable carbon source (usually potato starch) is added to stimulate the consumption of O2 by aerobic bacteria, generating anaerobic conditions in the soil slurry. Once anaerobic conditions are established, the degradation of the nitroaromatic compounds takes place. The use of potato starch as the external carbon source results in a large amount of starchy material remaining at the end of the treatment procedure. The resulting treated soil has a high oxygen demand due to the excess starchy material. This oxygen demand is detrimental to agricultural soils due to the rapid development of anaerobic conditions when the soil is wetted, such as after irrigation or rainfall. The excess oxygen demand also causes the treated soil to fail toxicity tests when Daphnia magna is used as a toxicity indicator due to the inability to keep enough oxygen in the test system for Daphnia magna to survive (unpublished results). In order to remove the excess oxygen left in the soil at the end of the treatment procedure, a second aerobic stage has been added to the treatment procedure. This paper describes the optimization of the amounts and type of external carbon added to minimize the time required for the aerobic utilization of excess carbon after the anaerobic stage is complete.

Materials and Methods Soils. Two soils were utilized in this research. The first is a sandy soil that was contaminated with TNT, RDX, and

VOL. 30, NO. 6, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

2021

HMX as result of the disposal of washings from munitions loading, handling, and dismantling at the Umatilla Army Ammunition Depot in Umatilla, OR. This soil is contaminated with 12 000 mg of TNT/kg of soil, 3000 mg of RDX/kg of soil, and 300 mg of HMX/kg of soil. The soil was sieved with a 10-mm sieve to remove gravel and stones. The sieved soil was stored at 4 °C until used. This soil is referred to as “Umatilla soil”. It contains no culturable organisms (32) and cannot be treated microbiologically without inoculation with a culture containing starch-degrading aerobes and nitroaromatic-degrading anaerobes (31). The second soil is a sandy loam from Ellensburg, WA. This soil was originally contaminated with Dinoseb and several other herbicides due to washing of crop-dusting equipment at an airfield. It was treated using the anaerobic treatment procedure until the Dinoseb and other herbicides were removed (28-30, 34). The aqueous phase was then decanted, and the soil was air-dried and stored at 4 °C until further use as an inoculum. This soil is referred to as “treated soil” and has been used successfully as an inoculum for the past 4 years. The air-drying and storage of the soil has only slightly affected its use as an inoculum. The microorganisms suspected of having important roles in the anaerobic metabolism of munitions compounds are anaerobic spore-forming organisms (Clostridia). These organisms survive desiccation and storage well. Preliminary studies on the enrichment of TNT-degrading organisms from the treated soil have revealed that 1 g of soil contains a population sufficient to enrich TNT-degrading organisms most of the time, while using 4 g of soil guarantees successful enrichment if the correct medium is used (35). The batch of treated soil used as inoculum for the enrichments reported above and the experiments reported here is the same and had been stored for 3-4 years. Experimental Design. All experiments were carried out in triplicate and have been reproduced at least once. The biological experiments were carried out in 500-mL Erlenmeyer flasks that received 4 g of Umatilla soil and 400 mL of 50 mM phosphate buffer containing 25 mM NH4Cl. An inoculum of 4 g of treated soil was added. External carbon was supplied as 1% (w/v) sodium acetate (Sigma), 1% (w/ v) insoluble starch (potato waste centrifuge cake, J. R. Simplot, Boise ID), 1% (w/v) soluble starch (Sigma), or 0.01, 0.1, 0.25, 0.5, 0.75, or 1% (w/v) glucose (Sigma). All cultures were incubated at 30 °C in the dark under static conditions. Aerobic incubations were carried out at room temperature on a rotary shaker at 100 rpm. Analytical Procedures. The concentrations of TNT, RDX, and the reduced TNT intermediates in the aqueous phase of the cultures were monitored as described previously (36). Samples of 0.5 mL were removed from the cultures at each time point and were centrifuged to remove suspended solids. A total of 10 µL of the supernatant fluid was injected onto an Alltech Alltima 5-µm RP C18, 250 × 2.1 mm i.d. column. The LC analyses was performed with a Hewlett-Packard HP 1090 Series II/M equipped with a DR-5 ternary solvent delivery system, variable-volume autoinjector, temperature-controlled autosampler, thermostatically controlled column compartment, and a diode array detection system. Separation of TNT, RDX, and the TNT reduction products was achieved using a gradient of 11 mM phosphate buffer and acetonitrile (36). The detector was set at 210 nm, and all peaks were scanned from 210 to 600 nm for compound identification and verification. All quantitation was performed using an external standard

2022

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 6, 1996

curve for each compound. Analytical standards were prepared from analytical grade chemicals. TNT was purchased from Chem Service (West Chester, PA). RDX was supplied by Darlene Bader (U.S. Athema). 2-Amino4,6-dinitrotoluene (2A), 4-amino-2,6-dinitrotoluene (4A), 2,4-diamino-6-nitrotoluene (24DA), 2,2′,6,6′-tetranitro-4,4′azoxytoluene, and 4,4′,6,6′ -tetranitro-2,2′-azoxytoluene were obtained through the generosity of Dr. R. J. Spanggord of SRI International, Menlo Park, CA. 2,6-Diamino-4nitrotoluene (26DA) was purchased from Aldrich. Redox measurements were performed using a Corning platinum combination redox electrode. Redox measurements were taken at the soil/water interface of each replicate culture. There was no sulfate present in the soil nor added to the reactors, so sulfate interference with the probe was not a problem. TOC measurements were performed using a Dohrman DC-80 TOC analyzer. Samples of 1 mL of the aqueous phase were centrifuged to remove solids, acidified with phosphoric acid and sparged with nitrogen to remove inorganic carbon before UV-promoted persulfate oxidation in the TOC analyzer. Desorption isotherms were carried out by placing various amounts of Umatilla soil in 100 mL of water or phosphate buffer. The concentrations of TNT in the aqueous phase were measured at different time points. Once the concentration in the aqueous phase stayed relatively constant the concentration of TNT in the soil was also determined. Statistical analyses were performed using the statistical software package SigmaStat (Jandel Scientific). One-way Anova, Dunnetts, and Student Newman-Keuls tests were performed to compare multiple means to a control or to each other.

Results and Discussion Sorption isotherms performed with Umatilla soil control flasks showed that the munitions compounds were not sorbed to the soil but were merely present as a part of the soil matrix. When the amount of soil used contained concentrations of TNT that produced concentrations of TNT in the aqueous phase that were lower than the solubility limit of TNT, there was no detectable TNT remaining in the soil. This suggests that the presence of TNT in the aqueous phase was governed by dissolution of the free product and the solubility of TNT in the aqueous phase, as opposed to reflecting an equilibrium with TNT sorbed to the sand. The amount of Umatilla soil used was set at 1% as this provided TNT in concentrations just at its solubility limit and allowed for a straightforward interpretation of the results. In controls where the inoculum or external carbon source was omitted from the reactor vessels, TNT degradation did not occur. The ability of cultures fed 1% (w/v) initial concentrations of glucose, soluble starch, insoluble starch, or acetate added as external carbon sources to consume oxygen and create anaerobic conditions necessary for removal of TNT and its reduced intermediates from contaminated soil was examined. The addition of glucose to the statically incubated soil cultures allowed the fastest reduction of the redox potential (Figure 1). The redox potential was measured at less than -400 mV after 4 days of incubation. This redox potential was maintained in these cultures for over 20 days. A complementary removal of TNT was observed in these cultures (Figure 2). The initial increase in TNT concentration over the first day of incubation, seen in this and other figures, is due to the dissolution of TNT from the soil into the aqueous phase of the cultures.

FIGURE 1. Redox potential in cultures of munitions-contaminated soil inoculated with treated soil and fed to 1% (w/v) initial concentrations of 2 insoluble starch, 9 soluble starch, 1 acetate, and b glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of redox potential measurements in triplicate reactors. The error bars indicate one standard deviation.

FIGURE 2. TNT concentration in cultures of munitions-contaminated soil inoculated with treated soil and fed to 1% (w/v) initial concentrations of 2 insoluble starch, 9 soluble starch, 1 acetate, and b glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of TNT concentrations in the aqueous phase in triplicate reactors. The error bars indicate one standard deviation.

The redox potential in the cultures fed insoluble starch was reduced to below 50 mV within 2 days and was maintained at this value for the entire incubation period. The presence of TNT and 4A in the cultures hold the redox potential at 50 mV due to their oxidized nature. In the cultures receiving insoluble starch as the external carbon source, the TNT was never completely removed (Figure 2); therefore, the redox potential in the cultures did not go below 50 mV. The apparent lack of ability to completely remove TNT in these cultures was investigated further and was discovered to be due to an inhibitory effect of the accumulation of 4-amino-2,6-dinitrotoluene (manuscript in preparation). In all cases when TNT was degraded, the production of 4-amino-2,6-dinitrotoluene and then 2,4diamino-6-nitrotoluene was observed. These intermediates were eventually removed in all cultures except when the redox potential increased to above -100 mV. The redox potential in the cultures fed acetate or soluble starch was not reduced to values much below 200 mV (Figure 1) nor was TNT removal accomplished in these cultures (Figure 2). The data for the TNT concentration for acetate cultures are identical to the data from isotherm and control studies. The TNT initially dissolves into the

aqueous phase, this is essentially complete by 2 days of incubation. After this period, the TNT concentration remains relatively constant when biological activity is absent. In these cultures as well as in isotherm and control cultures, the production and accumulation of the reduction intermediates were not detected. The inability of acetate to act as an electron donor for oxygen removal was unexpected. It is possible that TNT acts as an inhibitor of respiration, which has been observed for other nitroaromatic compounds such as Dinoseb and dinitro-o-cresol. Since acetate is a nonfermentable substrate, inhibition of respiration would prevent growth and oxygen utilization by these organisms. It is also possible that TNT inhibits either the TCA cycle or the glyoxylate shunt, which are the pathways required for organisms to grow on acetate. This inhibition may not effect growth and oxygen utilization in cultures fed glucose or starch because these substrates are fermentable, thus organisms can increase in number to the point where they can overcome the inhibition by TNT and utilize oxygen. The lack of growth and oxygen utilization in the cultures fed soluble starch may be due to a lack of starch-degrading organisms in the inoculum used. In contrast, the insoluble starch contains large numbers of starch degrading aerobes (28, 37). The concentrations of TNT and its metabolic intermediates over the time course of the incubation in the cultures fed glucose at 1% (w/v) are presented in Figure 3. The reduction of TNT to 4-amino-2,6-dinitrotoluene and 2,4diamino-6-nitrotoluene as well as the removal of these compounds was complete by 14 days (in other experiments the anaerobic phase was complete in as little as 8 days). This is an improvement of 10 days in the time required for the anaerobic stage over that presented by Funk et al. (31) in which the anaerobic stage took 24 days to reach this point. The initial amount of soil contained enough TNT to provide an aqueous phase concentration of approximately 0.53 mM. This was never observed in biological cultures due to the degradation of TNT occurring as it was dissolving into the aqueous phase. Higher concentrations of the intermediates are observed than TNT because they are degraded much more slowly than the TNT. The 2,4diamino-6-nitrotoluene accumulates to a higher concentration than the 4-amino-2,6-dinitrotoluene because it is degraded slower than 4-amino-2,6-dinitrotoluene. The 4-amino-2,6-dinitrotoluene accumulates to higher concentrations than the TNT because it is degraded more slowly than the TNT. A fungus was observed growing on the surface of the cultures reported in the work by Funk et al. (31). This was not observed in the present work. In the present work an aerobic stage was added after 25 days of static incubation in order to act as a polishing step to remove excess oxygen demand remaining from the glucose added and fermentation products of TNT. TOC in the culture supernatants was analyzed as an indication of the amount of organic carbon remaining at each time period. This does not provide information concerning the specific chemicals present in the cultures, but gives a general impression of the amount of organic carbon, which can be related to oxygen demand, remaining in the cultures at any time point. The initial TOC of 1% glucose solution should be 360 mM. The TOC left after the anaerobic stage was 175 mM, thus 185 mM organic carbon was used to create and maintain the anaerobic conditions in the reactor. The aerobic stage

VOL. 30, NO. 6, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

2023

FIGURE 3. Time course analysis of TNT, its metabolites, and TOC in cultures of munitions-contaminated soil inoculated with treated soil and fed 1% (w/v) of glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of triplicate analyses of 4 TNT, 9 RDX, ( 4-amino-2,6-dinitrotoluene, " 2,4-diamino-6-nitrotoluene, and b TOC. An aerobic stage was initiated after 25 days of anaerobic static incubation. The error bars indicate one standard deviation.

required an extra 50-55 days to remove the majority of the carbon present. This renders the aerobic polishing step as an uneconomical addition to the treatment procedure. The reduction of the redox potential and the removal of TNT and its metabolic intermediates from the cultures fed 1% (w/v) glucose occurred very rapidly. There was also 2100 mg/L (175 mM) TOC left in these cultures at the end of the anaerobic incubation. This suggests that the glucose had been added in excess. Experiments were conducted to determine whether smaller amounts of glucose could be added and still provide enough carbon for creation and maintenance of anaerobic conditions in the statically incubated soil slurry cultures. Figure 4 presents the redox potentials measured during the incubation of munitionscontaminated soil cultures fed various amounts of glucose. The cultures fed 1%, 0.75%, 0.5%, and 0.25% all reduced the redox potential to levels near or below -400 mV and maintained this level. Results from the cultures fed 0.5 and 0.75% glucose were not statistically different (P ) 0.05) from those fed 1% glucose, so they were not included in the figure. Redox potentials of -200 to -300 mV were observed in cultures fed 0.1% while the cultures fed 0.01% glucose reduced the redox potential only to -100 and could not maintain this redox potential. Figure 5 presents the TNT concentrations in these cultures. The use of 1% and 0.25% glucose as an external carbon source allowed the rapid and complete removal of TNT from the cultures. Results from cultures fed 0.75% and 0.5% glucose were not statistically different (P ) 0.05) from those presented for 1% and 0.25% glucose. In cultures fed 0.1% glucose, an initial rapid reduction in the TNT concentration was followed by an increase in TNT concentration. This resulted in significantly greater amounts of TNT in these cultures than in cultures fed greater amounts of glucose. The cultures fed 0.01% glucose were not capable of totally removing the TNT from the aqueous phase.

2024

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 6, 1996

FIGURE 4. Redox potential in cultures of munitions-contaminated soil inoculated with treated soil and fed glucose to initial concentrations of 2 0.01% (w/v), 1 0.1% (w/v), 9 0.25% (w/v), and ` 1% (w/v) in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of redox potential measurements in triplicate reactors. The error bars indicate one standard deviation.

The results of TOC analyses of the cultures fed decreasing levels of glucose are presented in Figure 6. TOC removal occurred at about the same rate in all of the cultures, independent of the TOC level present until TOC levels of 100 mg/L were observed. This indicates that the oxygen

FIGURE 5. TNT concentrations in cultures of munitions-contaminated soil inoculated with treated soil and fed glucose to initial concentrations of 2 0.01% (w/v), 1 0.1% (w/v), 9 0.25% (w/v), and ` 1% (w/v) in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of TNT concentrations in the supernatants of triplicate reactors. The error bars indicate one standard deviation.

FIGURE 7. Time course analysis of TNT, its metabolites, and TOC in cultures of munitions-contaminated soil inoculated with treated soil and fed 0.25% (w/v) of glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of triplicate analyses of 4 TNT, 9 RDX, ( 4-amino-2,6-dinitrotoluene, " 2,4-diamino-6nitrotoluene, and b TOC. An aerobic stage was initiated after 14 days of anaerobic static incubation. The error bars indicate one standard deviation.

of cultures fed 0.25% glucose are presented in Figure 7. TNT, RDX, and the TNT reduction products were removed by day 14, and an aerobic stage instituted on day 15 allowed the reduction of the TOC from approximately 400 mg/L (33mM) to 30 mg/L (2.5 mM) in 7 days. The whole procedure required 21 days of incubation. Past optimization experiments had resulted in an estimated 24 days time for completion of the anaerobic stage alone.

Acknowledgments This research was funded through a cooperative agreement with the U.S. Environmental Protection Agency’s Environmental Research Laboratory in Athens, GA.

Literature Cited

FIGURE 6. TOC in cultures of munitions-contaminated soil inoculated with treated soil in a 50 mM phosphate buffer solution with 25 mM NH4Cl and fed glucose to initial concentrations as indicated in figure. Data points represent the average of TOC measurements in triplicate reactors. The error bars indicate one standard deviation.

supply was the limiting factor. Cultures fed 0.25% glucose had removed the TOC present by day 21 (7 days aerobic incubation) leaving approximately 30 mg/L TOC residual. A concentration of 0.25% glucose was chosen as the optimum amount of external glucose to add since this was the lowest concentration of glucose fed to cultures that resulted in both a rapid reduction of the redox potential and TNT removal and the minimal time required for the aerobic stage. The results of the HPLC and TOC analysis

(1) Greene, B.; Kaplan, D. L.; Kaplan, A. M. Degradation of Pink Water Compounds in SoilsTNT, RDX, HMX; Technical Report NATICK/TR-85/046; United States Army Natick Research and Development Center: Natick, MA, 1985. (2) Kayser, E. G.; Burlinson, N. E. J. Energ. Mater. 1988, 6, 45. (3) Higson, F. K. Adv. Appl. Microbiol. 1992, 37, 1. (4) Kaplan, D. L.; Ross, E.; Emerson, D.; LeDoux, R.; Mayer, J.; Kaplan, A. M. Effects of Environmental Factors on the Transformation of 2,4,6-Trinitrotoluene in Soils; Technical Report NATICK/TR-85/ 052; United States Army Natick Research and Development Center: Natick MA, 1985. (5) Bradley, P. M.; Chapelle, F. H.; Landmeyer, J. E.; Schumacher, J. G. Appl. Environ. Microbiol. 1994, 60, 2170. (6) Spanggord, R. J.; Mortelmans, K. E.; Griffin, A. F.; Simmon, V. F. Environ. Mutagen. 1982, 4, 163. (7) Won, W. D.; DiSalvo, L. H.; Ng, J. Appl. Environ. Microbiol. 1976, 31, 576. (8) Palazzo, A. J.; Leggett, D. C. J. Environ. Qual. 1986, 15, 49. (9) Smock, L. A.; Stoneburner, D. L.; Clark, J. R. Water Res. 1976, 10, 537. (10) Degani, J. G. Trans Am. Fish. Soc. 1943, 73, 45. (11) Klausmeier, R. E.; Osmon, J. L.; Walls, D. R. Dev. Ind. Microbiol. 1973, 15, 309. (12) Amerkhanova, N. N.; Naumova, R. P. Sb. Aspir. Rab.-Kazan. Gos. Univ. im. V. I. Ul’yanova-Lenina, Estestv. Nauki 1975, 147. (13) Bumpus, J. A.; Tatarko, M. Curr. Microbiol. 1994, 28, 185. (14) Michels, J.; Gottschalk, G. Appl. Environ. Microbiol. 1994, 60, 187. (15) Spiker, J. K.; Crawford, D. L.; Crawford, R. L. Appl. Environ. Microbiol. 1992, 58, 3199. (16) Bradley, P. M.; Chapelle, F. H. Environ. Sci. Technol. 1995, 29, 802. (17) Kaplan, D. L. Curr. Opin. Biotechnol. 1992, 3, 253. (18) Walker, J. E.; Kaplan, D. L. Biodegradation 1992, 3, 369.

VOL. 30, NO. 6, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

2025

(19) Gorontzy, T.; Drzyzga, O.; Kahl, M. W.; Bruns-Nagel, D.; Breitung, J.; von Loew, E.; Blotevogel, K. H. Crit. Rev. Microbiol. 1994, 204, 265. (20) Kaplan, D. L. In Biotechnology and Biodegradation; Kamely, D., Chakrabarty, A., Omenn, G. S., Eds.; Advances in Applied Biotechnology Series; Portfolio Publishing Company: Woodlands, TX, 1990; pp 155-182. (21) Preuss, A.; Fimpel, J.; Diekert, G. Arch. Microbiol. 1993, 159, 345. (22) Boopathy, R.; Wilson, M.; Kulpa, C. F. Abstracts of Papers, 92nd General Meeting of the American Society for Microbiology, New Orleans, LA, 1992; Q143. (23) Boopathy, R.; Kulpa, C. F. Curr. Microbiol. 1992, 25, 235. (24) Boopathy, R.; Wilson, M.; Kulpa, C. F. Water Environ. Res. 1993, 65, 271. (25) Boopathy, R.; Kulpa, C. F.; Wilson, M. Appl. Microbiol. 1993, 39, 270. (26) Roberts, D. J.; Funk, S. B.; Korus, R. A. Abstracts of Papers, 92nd General Meeting of the American Society for Microbiology, New Orleans, LA, 1992; Q136. (27) Roberts, D. J.; Crawford, D. L. Abstracts of Papers, 91st General Meeting of the American Society for Microbiology, Dallas, TX, 1991; Q160. (28) Kaake, R. H.; Roberts, D. J.; Stevens, T. O.; Crawford, R. L.; Crawford, D. L. Appl. Environ. Microbiol. 1992, 58, 1683. (29) Roberts, D. J.; Kaake, R. H.; Funk, S. B.; Crawford, D. L.; Crawford, R. L. In Biotreatment of Industrial and Hazardous Wastes; Gealt,

2026

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 6, 1996

M., Levin, M., Eds.; McGraw Hill: New York, 1992; pp 219-244. (30) Roberts, D. J.; Kaake, R. H.; Funk, S. B.; Crawford, D. L.; Crawford, R. L. Appl. Biochem. Biotechnol. 1993, 39, 781. (31) Funk, S. B.; Roberts, D. J.; Crawford, D. L.; Crawford, R. L. Appl. Environ. Microbiol. 1993; 59, 2171. (32) Funk, S. B.; Crawford, D. L.; Roberts, D. J.; Crawford, R. L. In Bioremediation of Pollutants in Soil and Water; ASTM STP 1235; Schepart, B. S., Ed.; American Society for Testing and Materials: Philadelphia, 1994. (33) Funk, S. B.; Crawford, D. L.; Crawford, R. L.; Mead, G.; DavisHoover, W. Appl. Biochem. Biotechnol. 1994, 51/52, 625. (34) Kitts, C. L.; Cunningham, D. P.; Unkefer, P. J. Appl. Environ. Microbiol. 1994, 60, 4608. (35) Roberts, D. J.; Pendharkar, S. In Bioremediation of Recalcitrant Organics; Hinchee, R. E., Anderson, D. B., Hoeppel, R. E., Eds.; Batelle Press: Columbus, OH 1995; pp 273-280. (36) Ahmad, F.; Roberts, D. J. J. Chromatogr. 1995. 693, 167. (37) Stevens, T. O. Ph.D. Dissertation, University of Idaho, 1989.

Received for review October 31, 1995. Revised manuscript received February 26, 1996. Accepted February 27, 1996.X ES950814T X

Abstract published in Advance ACS Abstracts, May 1, 1996.