Biofiltration and Inhibitory Interactions of Gaseous Benzene

They often coexist and are released into the environment (1, 2). ... obtained by adjusting the injection rate of the solution and the flow rate of the...
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Environ. Sci. Technol. 2006, 40, 3089-3094

Biofiltration and Inhibitory Interactions of Gaseous Benzene, Toluene, Xylene, and Methyl tert-Butyl Ether EUN-HWA SHIM,† JAISOO KIM,† K Y U N G - S U K C H O , * ,† A N D HEE WOOK RYU‡ Department of Environmental Science and Engineering, Ewha Womans University, Seoul 120-750, South Korea, and Department of Chemical and Environmental Engineering, Soongsil University, Seoul 156-743, South Korea

This study evaluated the individual and combined removal capacities of benzene, toluene, and xylene (B, T, and X) in the presence and absence of methyl tert-butyl ether (MTBE) in a polyurethane biofilter inoculated with a BTXdegrading microbial consortium, and further examined their interactive effects in various mixtures. In addition, Polymerase chain reaction-denaturing gradient gel electrophoresis and phylogenetic analysis of 16S rRNA gene sequences were used to compare the microbial community structures found in biofilters exposed to the various gases and gas mixtures. The maximum individual elimination capacities (MECs) of B, T, and X were 200, 238, and 400 g m-3 h-1, respectively. There was no significant elimination of MTBE alone. Addition of MTBE decreased the MECs of B,T, and X to 75, 100, and 300 g m-3 h-1, respectively, indicating that benzene was most strongly inhibited by MTBE. When the three gases were mixed (B + T + X), the removal capacities of individual B, T, and X were 50, 90, and 200 g m-3 h-1, respectively. These capacities decreased to 40, 50, and 100 g m-3 h-1 when MTBE was added to the mix. The MEC of the three-gas mixture (B + T + X) was 340 g m-3 h-1, and that of the fourgas mixture was 200 g m-3 h-1. Although MTBE alone was not degraded by the biofilter, it could be co-metabolically degraded in the presence of toluene, benzene, or xylene with the MECs of 34, 23, and 14 g m-3 h-1, respectively. The microbial community structure analysis revealed that two large groups could be distinguished based on the presence or absence of MTBE, and many of the dominant bacteria in the consortia were closely related to bacteria isolated from aromatic hydrocarbon-contaminated sites and/ or oil wastewaters. These findings provide important new insights into biofiltration and may be used to improve the rational design of biofilters for remediation of petroleum gas-contaminated airstreams according to composition types of mixed gases.

1. Introduction Benzene, toluene, and xylene (BTX) are volatile simple aromatic hydrocarbons commonly found in crude petroleum * Corresponding author phone: 82-2-3277-2393; fax: 82-2-3275; e-mail: [email protected]. † Ewha Womans University. ‡ Soongsil University. 10.1021/es052099l CCC: $33.50 Published on Web 04/01/2006

 2006 American Chemical Society

and petroleum products such as gasoline, and methyl tertbutyl ether (MTBE) is a gasoline additive commonly used to reduce knocking and boost octane levels. They often coexist and are released into the environment (1, 2). Studies have shown that the presence of BTX compounds could either stimulate the biodegradation of other BTX compounds through cometabolism, or inhibit such degradation through competitive inhibition, diauxie, or catabolic repression (3). Studies have shown that MTBE has negligible and/or inhibitory effects on the biodegradation of BTX gases (4), while the presence of BTX had either negligible (5) or inhibitory effects (1) on MTBE degradation, as reported that no MTBE cometabolism occurred with BTX (6). Further studies on substrate interactions between BTX and MTBE, as well as shifts in bacterial community compositions as reported before (7), are necessary to facilitate the environmental remediation of BTX and MTBE. Biofiltration has been successfully applied for the treatment of volatile organic compounds (VOC) such as BTX and MTBE; this technique has proven cost-effective and environmentally friendly. Several studies have demonstrated that biofilters may be used for remediation of petroleum (gasoline) and BTX compounds (8, 9). However, since various waste gases are often simultaneously emitted from a single source, it is important to examine the effect of mixed gases on the removal efficiency of a biofilter. To facilitate rational biofilter design by clarifying the interrelationships of B, T, X, and MTBE during gaseous biodegradation, this study examined the elimination capacities of individual BTX and MTBE in single or mixed gases, tested the interactions of these gases, and used 16S rDNA Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing to analyze changes in microbial community structure in response to changes in gas composition.

2. Materials and Methods 2.1. Packing Material and Inoculums. The utilized filter packing material consisted of cubic polyurethane foam cubes (Seilsponge, Korea) with a dimension of 1.5 × 1.5 × 1.5 cm. The bulk density, water holding capacity, porosity, average pore size, and surface area of the material were 0.015 g‚cm-3, 57 g-H2O‚g-1, 98.8%, 0.8 mm, and 76.81 m2‚g-1, respectively (9). The BTX degradable culture, obtained from activated sludge in a wastewater treatment plant, was incubated in 5 L LB medium (10 g L-1 tryptone, 5 g L-1 yeast extract, 10 g L-1 NaCl) at room temperature for about 24 h, and then inoculated to the filter material. The medium used in the biofilter was the modified Bushnell-Haas (BH) medium, which contained (per liter) 0.409 g MgSO4‚7H2O, 0.0265 g CaCl2‚2H2O, 1 g KH2PO4, 1 g NH4NO3, 6 g Na2HPO4‚12H2O, 0.0833 g FeCl3‚6H2O, and 1 mL trace elements. The pH of the medium was adjusted from 6.5 to 7.5 by addition of CaCO3. 2.2. Biofilter Setup and Experimental Conditions. The laboratory-scale biofilter used in this study was made of a square acrylic resin column (150 × 150 × 650 mm), and consisted of a drain storage tank (150 × 150 × 180 mm), two biofilter-beds (each 150 × 150 × 200 mm), and a liquid distributor. Each biofilter bed was packed with 0.2 m of isolate-bound filter material. A stainless steel screen (150 × 150 mm) was placed on the bottom of each filter bed to support the filter material. Compressed air was passed through a volatilization chamber before it entered the biofilter. The chamber was connected to the biofilter with a liquid injection system consisting of a peristaltic pump and a liquid BTX and MTBE VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Experimental Conditions of Biofiltrationa Inlet concentration (µl L-1)

gas benzene (B) toluene (T) xylene (X) benzene + MTBE (B + M) toluene + MTBE (T + M) xylene + MTBE (X + M) benzene + Toluene + Xylene (B + T +X) Benzene + Toluene + Xylene + MTBE (B+T+X+M) MTBE (M)

340; 670; 1090; 1540 460; 870; 1350 680; 960; 2530 170 + 150; 360 + 360; 640 + 520 130 + 170; 380 + 320; 580 + 520 130 + 130; 610 + 310; 1100 + 490; 1830 + 640 150 + 120 + 30; 290 + 270 + 340; 480 + 510 + 750; 710 + 760 + 1230; 1030 + 1110 + 1870 70 + 50 + 10 + 60; 180 + 180 + 170 + 140; 310 + 330 + 450 + 230; 430 + 480 + 670 + 330; 550 + 610 + 880 + 422 440

a

The operation times for all the treatments were around 3 or 4 days after 5 weeks stabilization, and the baseline condition with BTX mixture was restored along with re-inoculation of the newly enriched culture before each chemical condition started.

storage bottle. BTX and MTBE gases were produced by injecting pure liquid BTX and MTBE into the volatilization chamber air stream using a peristaltic pump (flow rate, 0.01∼0.1 mL min-1; M930, Younglin Co., Ltd, Korea) (9). The required concentration of gases flowing into the biofilter was obtained by adjusting the injection rate of the solution and the flow rate of the air stream. The biofilter was first acclimatized for one week with an inlet BTX mixture (∼200 µL L-1 benzene, ∼200 µL L-1 toluene, and ∼350 µL L-1 xylene) at a constant flow rate (0.02 mL min-1) and a space velocity (SV) of 50 h-1 to establish steady-state conditions. The BTX and MTBE removal experiments focused on changes of inlet concentrations (summarized in Table 1) at a fixed SV of 50 h-1. For experiments, ∼250 ppmv of benzene at a SV of 50 h-1 was supplied to the acclimatized biofilter, and the outlet concentration was monitored until it remained constant for 30 min. Once the outlet concentration was constant, the inlet concentration was increased stepwise to 700, 1000, and 1500 µL L-1, and the removal capacities were estimated at each step. To examine whether the biofiltration capability remained constant after sampling, mixed BTX gases were resupplied. Similarly, the elimination capacities of single gases (toluene and xylene), binary mixtures (B + M, T + M, and X + M), a tertiary mixture (B + T + X), and a quaternary mixture (B + T + X + M) were examined. For maintenance of the biofilter (maintenance of essential mineral salts and moisture), 2.5 L of BH medium was sprayed on the top of the biofilter for 0.5 min every 5 h by a circulating pump. The BH medium in the drainage was replaced weekly. 2.3. Analytical Methods. The BTX and MTBE gases were collected from the biofilter using 1 mL gastight syringes (Hamilton series no. 1001; Hamilton Co., NV) equipped with Teflon Minnert fittings. Gases were analyzed with a gas chromatograph (M600D; Younglin Co., Korea) equipped with a Supelcowax 10TM column (30 m, 0.32 mm, and 0.25 µm; HP, U.S.A.) and a flame ionization detector. N2 was used as the carrier gas at a flow rate of 1.2 mL min-1. The temperatures of the oven, injector, and detector were fixed at 100, 230, and 230 °C, respectively. 2.4. Microbial Community Structure Analysis. Polymerase chain reaction and denaturing gradient gel electrophoresis of 16S rDNA fragments were used to analyze the bacterial community structure in the biofilter following exposure to the various gases. At the termination of each experiment, ∼0.3 g of bacterial cells were harvested from the biofilter and centrifuged, and genomic DNA was extracted using the BIO101 FastDNA SPIN KIT for soil (Q-Biogene, CA). PCR was used to amplify a 177 base pair portion of the 16S rDNA using primers 341FGC and 518R (10). The PCR amplifications were carried out as mentioned in the former research (11). The amplification conditions consisted of an initial denaturation of 93 °C for 2 min, followed by 35 cycles 3090

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of 92 °C for 1 min, 55 °C for 1 min and 68 °C for 45 s, followed by a final extension at 72 °C for 2 min (12). DGGE was performed as the previous research (10). The DGGE image was translated into the microbial community structure by comparison, using the GelCompar software (version 3.5; Applied Maths, Belgium) to perform unweighted pair group method with arithmetic mean (UPGMA) clustering using the Jaccard coefficient based on band position. 2.5. DNA Sequencing and Phylogenic Analysis. For sequencing, the previous research steps were followed (11). The obtained sequences were compared using the Basic Local Alignment Search Tool (BLAST) algorithm feature of the National Center for Biotechnology Information (NCBI) website. Sequences were initially aligned with each other and with manual adjustment using proBiosys version 1.0 operating under the default parameters (proBionic Corp., Daejeon, Korea).

3. Results 3.1. Acclimation of Individual BTX and MTBE. The removal tendency of benzene, toluene, and xylene was examined at a fixed SV of 50 h-1, based on the inlet and outlet gas concentrations when each gas was supplied individually to the biofilter (data not shown). After 15 days of biofilter operation, the outlet concentrations for each gas reached a constant, maintainable level, indicating that the biofilter was ready to be tested with different inlet concentrations. In contrast, MTBE was barely degraded in the biofilter over ∼40 days, indicating that the biofilter lacked efficient MTBE degraders (data not shown). 3.2. Elimination Capacities of BTX and MTBE in Single or Mixed Gases. Figure 1(a) shows the effects of MTBE, toluene, and xylene gases on benzene degradation, calculated in terms of elimination capacity. The inlet load was used to investigate the relationship between SV and inlet gas concentration, and to identify the main factors influencing the removal efficiency of BTX and MTBE. The load and the elimination capacity were calculated according the following equations:

Load ) (FCinlet)/V EC ) (F(Cinlet - Coutlet))/V where F is the flow rate (m3‚h-1), Cinlet is the inlet gas concentration (g‚m-3), Coutlet is the outlet gas concentration (g‚m-3), and V is the packing volume (m3). A comparison of the various benzene-degrading capacities revealed that the MECs of benzene were 200 g m-3 h-1 for the single gas, 75 g m-3 h-1 for binary gases (B + M), 50 g m-3 h-1 for the tertiary gas (B + T + X), and 40 g m-3 h-1 for the quaternary

FIGURE 1. Elimination capacities of B, T, X, and total BTX, versus the interactive effects of other gas(es). gas (B + T + X + M) (Figure 1a). The benzene elimination capacity in the single gas continuously increased according to the inlet load, while the benzene elimination capacities in the B + M, B + T + X, and B + T + X + M mixtures decreased as the number of mixed gases increased. Moreover, the benzene elimination capacities in the B + T + X and B + T + X + M mixtures decreased time-dependently at inlet loads of benzene above 100 and 60 g m-3 h-1, respectively. These findings indicate that the addition of MTBE, toluene, and xylene inhibited benzene degradation with increasing severity as the number of mixed gases increased. The effects of MTBE, benzene, and xylene on toluene degradation were then examined in a similar manner (Figure 1b). The MECs of toluene in the biofilter were 238, 100, 90, and 50 g m-3 h-1 for the single gas, binary gas (T + M), tertiary gas (B + T + X), and quaternary gas (B + T + X + M), respectively. The toluene elimination capacity declined at toluene inlet loads above 100 and 75 g m-3 h-1 of B + T + X and B + T + X + M, respectively. Comparison of the xylene removal capacity in the biofilter (Figure 1c) revealed that the MECs were 400, 300, 200, and 100 g m-3 h-1 for the single, binary (T + M), tertiary (B + T + X) and quaternary (B + T + X + M) gases, respectively. Similar to the above findings with benzene and toluene, the elimination capacity of toluene increased with inlet load in the cases of the single and binary gases. The xylene elimination capacities in the presence of the B + T + X and B + T + X + M mixtures decreased at loads of more than 400 and 150 g m-3 h-1, respectively. Examination of the elimination capacity of BTX gas and its inhibition by MTBE (Figure 1d) revealed that the elimination capacity of BTX increased to a maximum EC of 340 g m-3 h-1 in the absence of MTBE and of 200 g m-3 h-1 in the presence of MTBE. Biodegradation of MTBE was not observed

in biofilters exposed to BTX (data not shown), but did occur in the presence of toluene, benzene, or xylene alone, with maximum elimination capacities of 34, 23, and 14 g m-3 h-1, respectively (data not shown). Therefore, these findings indicate that xylene was most effectively degraded by the biofilter, while benzene was not degraded as effectively, and MTBE was hardly degraded at all. 3.3. Comparisons of Gas Interaction Indices. The gas interaction indices were based on the MECs and calculated according to the following equation (13):

gas interaction index ) (AB - A)/A where A ) MEC of compound A and AB ) MEC of compound A in the presence of compound B A positive index value indicated the presence of a synergistic interaction effect wherein additive compound(s) stimulated the degradation of individual BTXM or total BTX, while a negative index value indicated an inhibitory interaction effect. The size of the index value itself reflected the strength of the positive or negative interaction. The results revealed that MTBE and BTX gases inhibited B, T, and X degradation (Table 2). The interaction index of benzene was particularly strong, showing values of -0.63, -0.75, and -0.80 for the addition of MTBE, T + X, and T + X + M, respectively. Toluene showed slightly lower negative values, while degradation of xylene was not strongly inhibited by the other gases, showing interaction indices of -0.25, -0.5, and -0.75 for MTBE, B + T, and B + T + M, respectively. The inhibitory effect of MTBE on B, T, and X degradation (index -0.625 to -0.25) was lower VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. DGGE profiles of 16S rDNA sequences amplified from biofilter samples.

TABLE 2. Gas Interaction Indices Indicating the Effects of MTBE or Mixed Gases on the Biodegradation of B, T, or X no addition +M + two others (from BTX) + three others (from BTXM)

B

T

X

0.00 -0.63 -0.75 -0.80

0.00 -0.58 -0.62 -0.79

0.00 -0.25 -0.50 -0.75

than the inhibition of BTX by the two other gases (index -0.75 to -0.5). Interestingly, the inhibition of xylene degradation in the presence of benzene and toluene (XBT) was twice as strong in the presence of MTBE (XM), while inhibitions of benzene and toluene were increased by only 20% (BTX) and 10% (TBX), respectively, by the presence of MTBE. In the quaternary BTXM mixture, benzene was inhibited to a similar degree as seen in BTX, while toluene and xylene showed greater inhibition. This implies that benzene and toluene were more strongly inhibited by MTBE than xylene, regardless of whether the mixture was binary, tertiary, or quaternary. The inhibition of total gases (BTX) by MTBE showed a good fit with the average of the interaction index values (BM, TM, and XM). Collectively, these results revealed that MTBE, B, and T showed strong inhibitory effects on the degradation of individual BTX gases, and the individual BTX gases had a synergistic effect on degradation of MTBE. 3.4. Microbial Community Structure and Phylogenetic Analysis of DGGE Clones. Changes in the microbial community structure present the polyurethane biofilter according to substrate were revealed by 16S rDNA PCR-DGGE of microbial samples taken before and after inoculation (Figure 2). In general, the microbial community structure tended to become more simply distributed in the presence of individual or mixed gases. The most dominant strain in all the bacterial communities was BFE6, which may be an effective degrader of benzene,

toluene, xylene, and MTBE. While two other major strains, clone BFE7 and BFE11, seemed to endure and/or grow in all the gas conditions, BFE15 and BFE16 were likely to be inhibited by individual or mixed gas(es) but more greatly by MTBE (Figure 2). Clones BFE3 and BFE4 were found only in communities growing in the presence of T, X, T + M, X + M, and B + T + X + M, suggesting that these strains might degrade toluene and xylene, but not benzene. Clone BFE5 was absent from samples exposed to MTBE, whereas clone BFE13 was found exclusively in samples that were only found in the presence of MTBE, suggesting a MTBE-degrader. BFE10 and BFE12 were found in the presence of MTBE or xylene only; BFE10 might be an extremely low prevalence in the original sample due to no band. BFE14 was found only in samples grown in the presence of B and T; and BFE1, BFE2, BFE8, and BFE9 could not be distinguished clearly enough for analysis. Sequence analysis and BLAST searches revealed that 11 clones were classified as members of γ-proteobacteria, and five clones as members of Firmicutes. We found that many of the dominant bacteria in the consortia were closely related to bacteria isolated from aromatic hydrocarboncontaminated sites and/or oil wastewaters through the BLAST search (data not shown). The DGGE results were used to compare the similarities of the microbial community structure (Table 3) and prepare a UPGMA-typed dendogram showing the similarities obtained by Jaccard coefficient based on band position (data not shown). Biofilter communities exposed to B, T, and X clustered with those exposed to BTX and the original inoculum samples, showing similarities greater than 40%. Those grown in the presence of MTBE or mixes containing MTBE clustered together with more than 50% similarity. Two large branches (the B + T + X system and the MTBE system) had about 35% similarity each other, and showed about 30% similarity with communities grown in the presence of B + T + X + M.

4. Discussion Several other studies on microbial degradation concluded that xylene was the least degradable of the BTX compounds (3). However, the microbial consortia reported herein degraded xylene to a high degree and benzene to a lesser degree (Figure 1). This may indicate that the consortium in the present biofilter included microbes having a tol pathway, which recognizes xylene but not benzene as a substrate (14). Alternatively, this may reflect a difference in the effect of solubility on the present gas-based system versus the previously reported liquid systems, since biodegradability may be inversely correlated with solubility (benzene: 1780 mg L-1; toluene: 515 mg L-1; xylene: 198 mg L-1). It is possible that higher solubility (as seen in benzene, for example) may cause higher toxicity to microbes versus the less soluble toluene and xylene. Previous reports indicated that MTBE could be aerobically biodegraded by several bacterial species as a sole carbon and energy source (15). However, MTBE showed negligible

TABLE 3. Similarity (%) of DGGE Banding Patterns inoculum B T X MTBE B+M T+M X+M B+T+X B+T+X+M 3092

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inoculum

B

T

X

MTBE

B+M

T+M

X+M

B+T+X

B+T+X+M

100 44.4 40.7 44.8 33.3 27.3 33.3 35.7 35.7 19.4

100 45.8 39.3 50.0 34.5 26.9 34.6 40.0 36.0

100 46.2 39.1 35.7 39.1 36.0 47.8 17.9

100 50.0 50.0 50.0 52.0 58.3 42.3

100 50.0 57.9 68.4 33.3 34.8

100 44.0 52.0 15.2 19.4

100 68.4 28.0 24.0

100 30.8 50.0

100 37.5

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degradation in the present biofilter over approximately 15 days. This may indicate that, although the consortium used in this study was collected from highly oil-contaminated wastewater thought to contain BTEX and MTBE, this consortium might lack a strain such as PM1 (β-Proteobacterium), which is a well-known MTBE degrader (1). As no convincing evidence exists that MTBE biodegradation occurs rapidly in the field under natural conditions, these findings might indicate that MTBE biodegradation capabilities are not a ubiquitous response to MTBE contamination (1). Moreover, MTBE has a lower Henry’s constant (2), and thus a higher tendency to move from the gas to aqueous phase compared to BTEX compounds, suggesting that it may have a relatively high toxicity to a gas-removing biofilter system versus the other BTX gases. Interestingly, MTBE could be co-metabolized in the presence of benzene, toluene, or xylene (data not shown). This finding seems to suggest that MTBE could be metabolized by enzymes induced in the presence of benzene, toluene, or xylene via the tod or tol pathways. There have been only few prior reports regarding co-metabolism of MTBE with BTX. Garnier et al. (16) reported that aromatic compounds (benzene, toluene, and xylene) were unable to co-metabolize MTBE, while Koenigsberg et al. (17) found that benzene cometabolized MTBE. This discrepancy may be due to the relatively low levels of MTBE and additive gases examined in the prior studies based on the result in this study. Usually, the presence of toluene-acclimating bacteria enhance BEX (benzene, ethylbenzene, and xylene) degradation (13, 18), due to the similar chemical structures and biochemical enzyme pathways (tol or tod) utilized by the BTX gases (19). Based on this concept, the present mixture of gasoline mono-aromatic gases performed should have resulted in competition among the gases for enzyme sites, leading to toxicity, diauxie, or catabolic repression (20). Consistent with this notion, our results revealed that the presence of two other BTX gases prohibited the biodegradation of any individual BTX gas (Figure 1 and Table 2). However, this is inconsistent with previous findings that the presence of singly or mixed benzene, toluene, and xylene enhanced the overall degradation of individual BTX gases (3, 13). This discrepancy may be because of different types of bacteria having different degradation pathways or their low concentrations yielding no inhibitory effect. Indeed, our results revealed that degradation of benzene was most strongly inhibited in the tertiary BTX mixture, followed by toluene and xylene (Table 2). This contradicts previous findings that degradation of toluene was most strongly inhibited, followed by xylene and benzene (3). These discrepancies may be due to different types of bacteria having different degradation pathways as mentioned above, or different conditions such as slurry phase. In contrast to previous reports suggesting that the presence of MTBE did not affect the cell viability and activity of non-MTBE-degrading cultures (18), this study found that the degradation of benzene, toluene, and xylene was negatively influenced by the presence of MTBE. This discrepancy may be due to the relatively high concentration of MTBE used herein because of its solubility (43 000 mg L-1). The previous studies used MTBE at concentrations of 0-100 mg L-1 suggesting that cell growth and biodegradation of individual BTX gases is not inhibited by low concentrations of MTBE, but are inhibited once the MTBE concentration exceeds a threshold (Figure 1). Interestingly, the gas interaction indices (Table 2) showed that MTBE and BTX gases most negatively affected the degradation of benzene, with toluene showing an intermediate effect, and xylene the least impact. This order is inversely related to the number of methyl groups attached to the prevailing ring structure (benzene ) 0, toluene ) 1, and xylene

TABLE 4. Comparison of the Present Elimination Capacities of Individual or Total BTX Gases with Those Found in Previous Studies gas

elimination capacity (g m-3 h-1)

removal efficiency

reference

benzene

11 58 165

90% 90% 90%

3 22 this study

toluene

13 73.4 99 204

90% 94% 90% 90%

3 23 22 this study

xylene

17 50 73 278

90% 90% 90% 90%

3 22 23 this study

BTX

108 284.7 340

maximum EC maximum EC maximum EC

24 6 this study

) 2), perhaps indicating the presence of an enzymatic mechanism that favors binding to a molecule with more side branches. This is similar to the situation in which monooxygenase preferentially binds methane (21). Of the BTX gases, xylene had the highest MEC, and its biodegradation was inhibited to the lowest degree by the presence of the other gases. In these indices, the values for MTBE were larger than those of the other gases (all positive values compared to negative values of BTX gases), especially toluene (about +1.75) (data not shown). The MEC of MTBE alone was extremely low compared to those of the other gases, and increased in the presence of toluene or benzene, suggesting that the enzymes produced by toluene and benzene (the tod pathway) may more effectively cometabolize MTBE versus those triggered by xylene. Furthermore, the present findings suggest that the BTX mixture inhibited biofiltration of all applied gases by some microbial effects. The microbial community structure analysis (Figure 2) revealed three dominant bands at the same position in all samples, regardless of biofilter operation or substrate. This seems to indicate that these microorganisms might be capable of degrading both BTX and MTBE, or at least may have increased survival in the presence of MTBE. The main band, BFE7, might be a bacterium capable of degrading benzene, toluene, xylene, and MTBE. Band BFE5 appeared only in inoculums containing B, T, X, and BTX, while BFE13 was found only in samples grown in the presence of MTBE (M, B + M, T + M, X + M, B + T + X + M). This seems to indicate that growth and BTX degradation by BFE5 was inhibited in the presence of MTBE, while BFE13 may slightly degrade MTBE and was inhibited by one or more of the BTX gases. Clones BFE10 and BFE12 seemed to degrade MTBE and xylene but not benzene and toluene, while BFE3 and BFE4 appeared to degrade toluene and xylene regardless of MTBE. This may indicate that BFE3 and BFE4 oxidized alkylated compounds at the alkyl side chain via the same monooxygenase enzyme, while BFE10 and BFE12 oxidized alkylated compounds via different monooxygenase enzymes (20). Overall, the microbial community structures showed clear differences based on the presence or absence of MTBE (Figure 2). To evaluate the capability of the biofilter used in this study, the EC values were compared with those of previously reported biofilters (Table 4). The elimination capacities of the individual BTX gases found in this study were much higher (at least 2-3 times) than those of previously reported systems with similar removal efficiencies. The EC of total BTX in the present study was higher than that found other studies, but VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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was only slightly higher in terms of the single gases, indicating that the microbial consortium used in the present work may show substrate competition, toxicity or physicochemical hindrance. In sum, we herein present the first report of the effect of MTBE on BTX degradation and the interaction of BTX and MTBE in a biofilter. Future work will be required to examine these effects under other conditions with other substrates.

Acknowledgments This work was financially supported by the Korea Science and Engineering Foundation through the Advanced Environmental Biotechnology Research Center at Pohang University of Science and Technology (R11-2003-006).

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Received for review October 22, 2005. Revised manuscript received February 14, 2006. Accepted February 16, 2006. ES052099L