Environ. Sci. Techno/. 1995, 29, 802-806
Factors affecting Microbial 2,4,6 -Triaikuatoluene
Introduction
PAUL M. BRADLEY* AND F R A N C I S H . CHAPELLE US.Geological Survey, Water Resources Division, Stephenson Center, Suite 129, 720 Gracern Road, Columbia, South Carolina 29210- 7651 ~
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The influence of selected environmental factors on microbial TNT mineralization in soils collected from a TNT-contaminated site at Weldon Spring, MO, was examined using uniformly ring-labeled [14C]TNT. Microbial TNT mineralization was significantly inhibited by the addition of cellobiose and syringate. This response suggests that the indigenous microorganisms are capable of metabolizing TNT but preferentially utilize less recalcitrant substrates when available. The observed inhibition of TNT mineralization by TNT concentrations higher than 100 pmollkg of soil and by dry soil conditions suggests that toxic inhibition of microbial activity a t high TNT concentrations and the periodic drying of these soils have contributed to the long-term persistence of TNT at Weldon Spring. In comparison to aerobic microcosms, mineralization was inhibited in anaerobic microcosms and in microcosms with a headspace of air amended with oxygen, suggesting that a mosaic of aerobic and anaerobic conditions may optimize TNT degradation atthis site.
At many military sites throughout the United States, high concentrations of 2,4,6-trinitrotoluene (TNV are commonly found where explosives loading, handling, and packaging activities were concentrated in the past (1). TNT is an environmental concern because of its toxicity to fish (2,3), algal species (3, 4 ) , microorganisms (4, 3,and other organisms ( 4 ) . At present, incineration is the most effective technology for remediation of TNT-contaminated soil. However, because of the costs of soil excavation, transport, and incineration, this approach is expensive (6). A recent demonstration of TNT degradation by microorganisms indigenous to the soils at an inactive munitions plant near Weldon Spring, MO (7), indicates that in situ bioremediation may be a realistic alternative for cleanup of nitroaromatic contaminated sites. Complete removal of TNT from microcosms containing surface soil collected within an inactive munitions wash house area was achieved in 22 days at an initial dissolved TNT concentration of approximately 100 pmol/L. More important from a bioremediation point of view was the observation that a significant fraction of added ring-labeled [14C]TNTwas mineralized to 14C02 within 35 days. However, the continuing presence of TNT at the Weldon Spring site even though munitions activity was discontinued circa 1945 suggests that some combination of environmental factors is inhibiting microbial degradation at the site. Consequently, in order to effectively bioremediate TNT contamination in situ, an identification of these factors is first necessary. The purpose of the studies reported here was to evaluate the effects of carbon substrate availability, soil moisture content, TNT concentration, and oxygen condition on TNT mineralization by the microorganisms indigenous to explosives-contaminated soil at Weldon Spring. The results indicate that microbial TNT mineralization in this soil was favored under microaerobic conditions. TNT mineralization was inhibited by the addition of complex carbon substrates and by decreasing soil moisture. In addition, TNT concentrations in excess of 100 pmollkg of dry soil inhibited mineralization.
Methods TNTwas obtained from SRI Intemational (Menlo Park, CAI. Cellobiose and syringate were obtained from Aldrich Chemical Company (Milwaukee, WI). The purity of all standards was 99%or greater. Uniformlyring-labeled [14C]TNT (26.3 mcilmmol) was obtained from Du Pont (NEN Research Products, Boston, MA). The purity of the radiolabeled TNT was determined by thin-layer chromatography and high-performance liquid chromatography (HPLC)and found to be greater than 99%. TNT mineralization was investigated using surface soil (red tank soil) collected within an inactive munitions wash house area at Weldon Spring, MO. The soil characteristics at the Weldon Spring site have been described in detail elsewhere (7).Although the wash house area had a history of high levels of TNT contamination (up to 357 mmol/kg * Corresponding author;Telephone: 803-750-6100;Fax: 803-7506181.
802 1 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3, 1995
This article not subject t o U S . copyright. Published 1995 by the American Chemical Society.
of drysoil), HPLC analyses indicated that the concentration of TNT in the collected soil was less than 1pmol/kg. Soil samples were collected with hand tools, which were rinsed with alcohol and flame-sterilized prior to use. Samples were stored at 4 "C prior to beginning microcosm studies. The effect of carbon substrate addition on aerobic TNT mineralization was investigated using cellobiose and syringate as carbon supplements. Microcosms consisted of 4 g of moist soil (3 g dry weight) and 2 mL of sterile TNT solution in sterile 30-mL serum vials. Microcosms were prepared aerobically and sealed with thick butyl rubber stopper/base-trap assemblies as described previously (7). The microcosm TNT concentration was approximately 200 pmol/kg of which approximately 2.4 x lo5 dpm (disintegrations per minute) per vial was [14C]TNT. Cellobiose or syringate was added to final concentrations of 20, 200, or 2000 pmollkg. Experimental microcosms were prepared in triplicate for each TNT concentration. Duplicate abiological controls were prepared by sterilizingthe microcosms (5 mmol/L HgC1,; autoclaved at 121 "C for 1 h) prior to addition of the test substrate. Soil microcosms for studying the effects of soil moisture on aerobic and anaerobic TNT mineralization were prepared by placing 3 g of dry soil in sterile 30-mL serum vials. The soil was dried at 35 "C for 5 days to yield a moisture content of 7% (by weight). Up to 3 mL of deionized water was added as needed to give final soil moisture contents of 7% (no water added), 15%, 28%, and 52% (by weight). To distinguish the effects of the drying process from the effects of soil moisture, additional microcosms were prepared with freshly collected moist soil (28%by weight water content) or with moist soil and 2 mL of distilled water added (52%by weight water content). All microcosms were spiked with 20 nmol of TNT, of which approximately 2.4 x lo5 dpm per vial was [14C]TNT.Triplicate experimental microcosms and duplicate abiological controls were prepared for each treatment. The effects of TNT concentration on overall soil microbial metabolism were determined by measuring the rate of CO2 production in microcosms containing unlabeled TNT at concentrations up to 1000 pmollkg. Aerobic microcosms were created as described above. Additional microcosms were prepared using soil from an adjacent site (topsoil) with no detectable TNT contamination and no history of nitroaromatic exposure. Triplicate experimental microcosms and duplicate abiological controls were prepared for each treatment. COz production was quantified by thermal conductivity detection gas chromatography. Dissolved COz concentrations were estimated from Henry's law coefficients (8). Soil microcosms for studying the effects of TNT concentration on TNT mineralization were prepared as described above. Additional microcosms were created with a helium headspace or a headspace of air amended with oxygen to give a final headspace 0 2 concentration of 50% by volume. The treatment solution was a mixture of unlabeled TNT and [14C]TNTdiluted as needed to yield final microcosm concentrations ranging from 0.05 to 1000 pmollkg of total TNT. Triplicate experimental microcosms and duplicate abiological controls were prepared for each treatment. Microcosmswere incubated staticallyin the dark at room temperature. After 60 days, the microcosms containing labeled TNT were acidified with 1000 p L of 2 N HsP04. Evolved 14C02was collected by placing 300 p L of 3 N KOH
in the suspended base traps and shaking the acidified microcosms for 48 h. The 14C02recovered in the base solution was quantified by liquid scintillation counting. Because the radiolabeled purity of the [14C]TNT was determined to be 99%,TNT mineralization was considered significant only if the activity recovered in the base traps was at least 1%of the amount added initially. The total amount of COz evolved from TNT was estimated based on the ratio of labeled and unlabeled TNT initially added to the microcosms. The recovery efficiency of 14C02 in the sample material was determined using H14C03. Reported values were corrected for recovery efficiencies, the activity recovered at time zero, and the activitydetected in sterilized control vials. The activity associated with radiolabeled volatile intermediates other than CO2 was estimated using toluene as a trapping agent and found to be less than 1% of the activity recovered in the base traps. For all studies, statisticallysigniticant differences between treatment means were identified by analysis of variance and the StudentNewman-Keuls Multiple Comparison test (9). To facilitate a comparison of results between this and earlier studies, the fraction of TNT in the microcosms that was adsorbed to the soil was approximately 40% and 65% for red tank and topsoil microcosms, respectively (Bradley,unpublished data).
Results and Discussion Several recent studies have suggested various biological methods for the cleanup of TNT-contaminated soil (61013). The results of composting studies are encouraging, but the requirement for excavation and transport of the contaminated soil, addition of supplemental carbon, long incubation periods, and in some cases, inoculation with a suitable microbial population make this approach expensive (6). Moreover, the ultimate fate of TNT during composting of contaminated soils is not clear, but cleavage of the aromatic ring does not appear significant ( 11). Alternatively, Fernando et al. (13)have suggested that the white rot fungus Pseudomonas chrysosporium may be an effective agent for remediation of TNT-contaminated systems. P. chrysosporium has been shown to degrade TNT to COz at TNT concentrations of about 6pmollL (13). However,the utility of this species as an agent for bioremediation of heavily contaminated soils has been questioned (14). Finally, Funk et al. (6) proposed that an anaerobic bioslurry technology previously used to bioremediate soils contaminated with nitroaromatic herbicides may be a suitable alternative for remediation of soils contaminated with nitroaromatic munitions compounds. Using this technique, removal of TNT from contaminated soil was achieved in 4 days, but significant TNT mineralization was not reported (6). An effective in situ bioremediation system based on the activity of indigenous microorganisms potentially can avoid many ofthe costs associatedwith the approaches described above. In order for in situ bioremediation to be an effective alternative for the cleanup of TNT contaminated sites, however, it is necessary to identify those factors which influence microbial TNT degradation under the in situ conditions. Degradation of a xenobiotic compound can occur cometabolically or as the result of direct utilization of the compound as an energy source or as a carbon substrate for cell growth and metabolism. Because contaminant cometabolism requires an additional substrate for growth or energy production, this process may be stimulated by the VOL. 29, NO. 3, 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY 1803
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FIGURE 1. Effect of supplemental carbon concentrations on the mineralization of TNT in microcosms containing red tank soil. The initial TNT concentration in the microcosmswas 2lMpmoUkg. Datum points are means of triplicate microcosms k SO. No significant mineralization was observed in killed controls.
addition of a metabolizable carbon substrate. P. chrysosporium degraded TNT when ground corn cobs were added to soil as a nutrient (13). Osmon and Klausmeier (2) reported that TNT degradation occurred only in the presence of supplemental organic material and concluded that TNT degradation was co-metabolic. Similarly, TNT degradation by Pseudomonas-likeorganisms was enhanced in the presence of glucose or yeast extract (15). In contrast, others have reported microbial growth using TNT as the sole carbon source (16). In the present study, carbon substrate addition significantly inhibited TNT mineralization by red tank soil microorganisms. Previous studies have indicated that the addition of natural carbon substrates such as corn cobs (13) and other plant products (11)can stimulate microbial degradation of nitroaromatic compounds. In the present study, cellobiose and syringate were chosen as models of naturally occurring cellulose and lignin-type compounds, respectively. Because the addition of cellobiose and syringate increased the total amount of COz produced in the red tank microcosms (data not shown), the red tank microorganisms were capable of metabolizing both compounds. Rather than stimulating TNT degradation, however, the addition of 2000 pmollkg of cellobiose and syringate reduced TNT mineralization by 66% and 42%, respectively, compared with mineralization in microcosms amendedwithTNT alone (Figure 1). These results indicate that addition of complex carbon substrates is not a suitable approach for stimulating TNT mineralization at this site. During the summer, the red tank soil community is frequently subjected to long periods of dry weather. Because of the potential negative impact of low soil moisture on microbial degradation of TNT, we examined TNT mineralization at different soil moisture levels. At sample collection, the red tank soil was fully saturated and contained approximately 28% (by weight) water. Addition of water to aerobic or anaerobic microcosms which contained undisturbed soil did not significantly affect TNT mineralization (Figure2). However, soil drying significantly inhibited TNT mineralization under aerobic and anaerobic conditions. Soil drying completely inhibited TNT mineralization in anaerobic microcosms at each moisture level. TNT mineralization in aerobic microcosms, which were dried and then remoisturized, increased with soil moisture content. The maximum mineralization in microcosms 804 1 ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3, 1995
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FIGURE2. Effect of soil drying and soil moisture content on aerobic and anaerobic mineralization of TNT in microcosms containing red tank soil. Closed symbols denote microcosms containing moist soil adjusted as needed to give the final moisture content indicated. Open symbols denote microcosms containing soil dried to a 7% water content and adjusted as needed to give the final moisture content indicated. Datum points are means of triplicate microcosms fSD. No significant mineralization was observed in killed controls.
containing dried soil was observed in microcosms with standingwater (52%moisture content). The results indicate that the soil drying, which typically occurs during the summer at Weldon Spring, can negatively impact in situ biodegradation of TNT and may contribute to the persistence of TNT at Weldon Spring in spite of the presence of an indigenous microbial community capable of TNT degradation. The toxic effects of elevated TNT concentrations on microbial activity have been described previously (4, 5). TNT concentrations in excess of 220 pmollL prevented or severely inhibited the growth of Gram-positive bacteria, actinomycetes,yeast, and fungi, but most ofthese organisms grew at TNT concentrations less than 88 pmollL (5). TNT caused frameshift mutations in Salmonella typhimurium and inhibited growth at concentrations above 44 pmollL (4). In contrast, Klausmeier et al. (5)reported that many Gram-negative bacteria were capable of growth at TNT concentrations of 440 pmol/L. A similar decline in activity with increasing TNT concentration was observed in the current study in microcosms containing soil from the uncontaminated topsoil site (Figure 3). Concentrations of TNT as low as 1 pmollkg significantly inhibited microbial activity in this soil. However, significant CO2 production was apparent even at TNT concentrations up to 1000pmol/kg. Because this soil was collected at a site with no detectable TNT and no history of nitroaromatics contamination, this experiment examined the short-term effects of contaminant exposure on an unacclimated microbial community. These results indicate that, over the short term, exposure to even low (1 pmollkg or less) concentrations of TNT can be inhibitory to microbial communities. In contrast, TNT concentrations in the range of 1- 100 pmollkg stimulated activity in the red tank microcosms (Figure 3). Over the range of 0.1-lOpmol/kg ofTNT, COz production in these microcosms increased from background levels to near maximum. Addition of TNT to a concentration of 100 pmollkg did not Significantly reduce microbial activity in this sediment below the rate observed at 10 pmollkg. A TNT concentration of 1000 pmollkg completely inhibited microbial activity in this soil. A comparison of the response of the contaminated red tank
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FIGURE 3. Effect of TNT concentration on the net CO2 production rate in microcosms containing contaminated red tank soil or uncontaminatedtopsoil. Rates are expressed as a percentage of the maximum rate observed for each soil type. Datum points are means of triplicate microcosms f SD. No significant mineralization was observed in killed controls.
microcosms and the uncontaminated topsoil microcosms suggests that the lack of apparent toxicity of nitroaromatic concentrations as high as 100 pmollkg is attributable to acclimation of the red tank microbial communities to nitroaromatics contamination. The effect of TNT concentrations on TNT mineralization by red tank microorganisms was investigated in order (a) to determine if the elevated TNT concentrations typical of the highly contaminated soil at Weldon Spring have contributed to the persistence of TNT at the site and (b) to identify the level to which soil TNT concentrations should be adjusted to optimize microbial degradationin situ. Under aerobic conditions, the recovery of [14C]TNTactivity as 14C02 after a 60-day incubation period was approximately 11%in microcosms with TNT concentrations ranging from 0.05 to 100pmollkg (datanot shown). Severalinvestigators have used a radiolabeled substrate to examine TNT degradation by a variety of microorganisms (11,13,14,17, 181,but only the white rot fungus P. chrysosporium (13,14) and the microorganisms indigenous to Weldon Spring (7,
this study) have demonstrated significantTNT ring cleavage and mineralization. The recovery of the radiolabel was reduced to 5.5 k 1.6%at aTNT concentration of 250pmoll kg, 1.7 f0.1%at 500pmol/kg, and0 & 0% at lOOOpmol/kg. This response was similar to the inhibition of TNT mineralization in P. chrysosporium at TNT concentrations of 66 pmollL or more. The amount of TNT mineralized by the red tank microbial community increased with TNT concentration up to a concentration of approximately100pmol/kg (Figure 4). TNT mineralization did not vary significantly between 100 and 250 pmollkg of treatments; however, a TNT concentration of 500 pmollkg significantly inhibited mineralization. These results are consistent with the continued presence of TNT at Weldon Spring even though munitions activities were discontinued at the site circa 1945. Based on the present study, the elevated TNT concentrations frequently observed at this site would be expected to inhibit microbial TNT degradation. Microbial degradation of nitroaromatic compounds has been observed under aerobic and anaerobic conditions (2, 6, 7, 11, 13-15, 17-28). Aerobic degradation of 2,4dinitrotoluene, 2-nitrotoluene, 3-nitrotoluene, and 4-nitrotoluene via oxidative pathways has been described for environmental isolates of Pseudomonas sp. (27,29,30),but most investigators have reported degradation of TNT and DNT via reductive pathways under aerobic as well as anaerobicconditions (2,6, 7,11,13-15,17-26,28). Under both conditions, the para nitro group of TNT is typically reduced through nitroso and hydroxlamino intermediates to form aminodinitrotoluenes (6, 11, 15, 17, 18, 20, 21,25, 26). Under aerobic conditions, accumulation of unstable hydroxylamino intermediates can lead to the formation of azoxy dimers that are resistant to further degradation (6, 11,17,18,25,26). Because of the widely reported formation of azoxytetranitrotoluene compounds under aerobic conditions, some have concluded that efficient degradation of TNT requires anaerobic conditions (6). In spite of this evidence, the effect of aerobic vs anaerobic conditions on microbial TNT degradation remains controversial in large part because significant mineralization of TNT has been AIR
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TNT CONCENTRATION (pmoledkg) FIGURE 4. Effect of TNT concentretion on the mineralization of TNT in microcosms containing red tank soil incubated under anaerobic (helium), aerobic (air), or oxygen-amended conditions. Datum points are means of triplicate microcosms fSO. No significant mineralization was observed in killed controls.
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reported only in cultures incubated with an aerobic headspace (7, 13, 14). To clarify the effect of oxygen condition on microbial TNT degradation, mineralization of TNT by red tank microorganisms incubated with an aerobic headspace was compared with mineralization occurring under strict anaerobic conditions. Additional microcosms with a headspace of 02-amended air were created to determine the effect on TNT mineralization of an increase in the diffusive supply of 0 2 to the soil microbial community. For all TNT treatments examined in this study, the highest recovery of [14C]TNTactivity was observed in the microcosms that contained an air headspace (Figure 4). Under strict anaerobic conditions, the recovery of I4CO2was 1% or less. These results indicate that strict anaerobic conditions do not favor microbial degradation of TNT to C02 in this soil. It is unlikely, however, that the mineralization of TNT observed in the red tank microcosms was a strictly aerobic process. Under static culture conditions, like those maintained in this study, the microbial environment within saturated soils is predominantly anaerobic (7, 31). The stoichiometric conversion of TNT to the transient intermediates, 4-amino-2,6-dinitrotoluene and 2-amino-4,6dinitrotoluene, observed previously in red tank microcosms (7) was consistent with earlier reports of anaerobic degradation of nitroaromatic compounds (6, 22, 23). The fact that increasing the headspace 0 2 concentration and thereby increasing the diffusive flux of 0 2 to the soil microbial community significantly reduced TNT mineralization below that observed in microcosms with an unamended air headspace (Figure 4) provides further evidence that TNT mineralization by red tank microorganisms is favored under microaerobic conditions. Although TNT degradation products other than COz were not analyzed, the decrease in TNT mineralization observed in the 02amended microcosms may reflect a shift from complete degradation to COz toward formation of recalcitrant products like azoxytetranitrotoluene. Previously, Funk et al. (6) suggested that an efficient system for complete degradation of TNT to nonaromatic end products may require a combination of anaerobic and aerobic treatment. The present results, which are consistent with their conclusion, indicate that TNT mineralization is favored under heterogenous conditions where both anaerobic and aerobic metabolism are possible.
Acknowledgments This research was completed in cooperation with the U.S. Army Corps of Engineers, Kansas City District. We thank Judith C. Pennington of the US.Army Engineer Waterways Experiment Station for providing the [l4C1TNT.
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Received for review August 16, 1994. Revised manuscript received November 3, 1994. Accepted November 15, 1994.@
ES940521K @
Abstract published inAduunceACSAbsnucts,December 15,1994.
