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MTBE biodegradation and degrader microbial community dynamics in MTBE, BTEX, and heavy metal-contaminated water
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International Biodeterioration & Biodegradation 59 (2007) 97–102 www.elsevier.com/locate/ibiod
MTBE biodegradation and degrader microbial community dynamics in MTBE, BTEX, and heavy metal-contaminated water Chi-Wen Lina,, Hung-Chun Lina, Chi-Yung Laib a
Department of Environmental Engineering, Da-Yeh University, 112 Shan-Jiau Rd., Da-Tsuen, Changhua 515, Taiwan, ROC b Department of Biology, National Changhua University of Education, Changhua 515, Taiwan, ROC Received 12 March 2006; received in revised form 21 June 2006; accepted 31 July 2006 Available online 26 September 2006
Abstract The aim of this investigation was to explore microbial community changes under various environmental groundwater conditions (single substrate, mixed substrates, and the presence of heavy metals) and link the changes with simultaneously diminishing substrate concentration in the microcosms. Most microorganisms from environmental microcosms or wastewater treatment plants cannot be cultivated artificially. Capturing microbial community fingerprints, therefore, requires applying a molecular biological technique. By using SSCP profiles of PCR-amplified 16S rDNA genes, it was demonstrated that with the repeated addition of substrates during longterm acclimation, substrate-utilizing populations in a microcosm gradually increased to become the dominant constituent. Conversely, the presence of metals inhibited community development and differentiation. It was also shown that substrate degradation rates increased under co-substrate conditions, with substrate-degraders easily adapting to the environment and becoming the dominant bacteria, a phenomenon attributed to the propensity of the fittest species to outgrow their competitors when presented with suitable substrates. r 2006 Elsevier Ltd. All rights reserved. Keywords: 16S rDNA; Metal ions; Microbial community structure; Molecular profiling; Methyl tert-butyl ether
1. Introduction Methyl tertiary butyl ether (MTBE) is one of several fuel oxygenates added to gasoline to replace tetraethyl lead and reduce harmful tailpipe emissions. The present guideline limit established by the US Environmental Protection Agency (EPA) is 20–40 mg l1 for MTBE in drinking water (Jacobs et al., 2001). The EPA also classifies MTBE as a possible human carcinogen (Johnson et al., 2000). Numerous studies have demonstrated that MTBE can be biodegraded under aerobic or anaerobic conditions (Eweis et al., 1997; Mo et al., 1997; Steffan et al., 1997; Hanson et al., 1999; Bruns et al., 2001; Hristova et al., 2003). The MTBE biodegradation rate may further diminish in the presence of co-contaminants, such as benzene, ethylbenzene, toluene, and xylene (BTEX). However, little is known
Corresponding author. Tel.: +886 4 8511339; fax: +886 4 8511347.
E-mail address: [email protected] (C.-W. Lin). 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.08.002
about the microbial community structure during aerobic MTBE degradation in the presence of BTEX. The inhibitory effects on MTBE biodegradation have recently been investigated by several researchers. These studies have focused mainly on substrate inhibition (Park, 1999), by-product inhibition (Liu et al., 2001; Wilson et al., 2002) or competitive inhibition (Garnier et al., 1999; Sedran et al., 2002). However, little has been published on the effect of heavy metals on MTBE biodegradation. Many types of pollutants may coexist in a contaminated site. For instance, organic chemicals and heavy metals can appear simultaneously in a leaking oil tank. Reports by several researchers (Baldrian et al., 2000; Bruins et al., 2000) indicate that some heavy metals (Cu, Zn, Cd) produce toxicity and may also simultaneously affect organic contaminant biodegradation (Said and Lewis, 1991) owing to heavy metal accumulation in the microbial cells. Metal toxicity on microorganisms has been studied by growth inhibition, while heavy metal toxic effects on bacterial community structure have been studied to a
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lesser extent. To fully exploit MTBE bioremediation technology for field application, the biodegradation and microbial community structure in the presence of heavy metals must be better understood. Most microorganisms cannot be detected in soil and groundwater samples using cultivation-based methods (Amann et al., 1995; Ranjard et al., 2000). Conventional microorganism analyses cannot fully reveal the species and the resulting microbial community composition in the environment. Hence, it is necessary to utilize molecular approaches to provide clues to bacterial types involved in aerobic phenol degradation and determine the relationship between growth and decline among bacterial populations. Methods for exploring the microbial community dynamics during in situ bioremediation has recently received increased attention. Griffiths et al. (1999) employed a DNA–phospholipids fatty acid (PLFA) method to analyze the effects of varying substrate concentrations on a soil microbial community. Their findings indicated that, in the substrate range 0–125 mg ml1, lower substrate concentrations resulted in less change in community structure, as evidenced by high similarity indices (0.8–0.9) between preand post-incubation samples. Peter et al. (2000) demonstrated that compost microbial communities are dominated by a few species with the most favoured growth requirements. Hristova et al. (2001) dealt with molecular fingerprinting techniques and detected the MTBE-degrading strain PM1 by real-time TaqMan PCR. Deeb et al. (2001) showed the substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate. These authors have proposed some tools for determining whether certain degraders are present, and have shown inhibition by o-xylene. However, they did not address the changing composition of microbial communities. Zhang et al. (2005) studied a sequencing batch reactor (SBR) system using PCR–denaturing gradient gel electrophoresis (DGGE) and reported that the resident community reached a stable structure dominated by nine species after operation for 200 days. Single-strand-conformation polymorphism (SSCP) is one of the methods frequently used, being inexpensive and highly sensitive (Lee et al., 1996; Schwieger and Tebbe, 1998). In recent years, it has been applied extensively to detect mixed culture diversity in the environment. For instance, Stach et al. (2001) showed that SSCP microbial community analysis could be used to compare the efficiency of alternative methods for DNA extraction and purification. Balcke et al. (2004) used PCR–SSCP to evaluate microbial community changes under consecutive aerobic–anaerobic and various pH conditions. Dassonville et al. (2004) examined microbial dynamics in anaerobic soil slurry and analyzed their dependence on geochemical processes by using the SSCP technique. In this study MTBE biodegradation was analyzed in the presence of other contaminants in batch culture experiments. Some of the contaminants served as co-substrates while the others functioned as growth inhibitors. Then the
SSCP method was combined with batch culture experiments to examine the microbial population and community changes during degradation. 2. Materials and methods 2.1. Bacterial strains and batch microcosm experiments The bacterial cultures used in this study were obtained from a petrochemical wastewater treatment plant. They were initially grown in a liquid medium and adapted to the targeted compounds and working conditions before the biodegradation experiments. After a series of screening and isolation procedures, three pure cultures were identified as Ralstonia sp. (YABE411), Pseudomonas sp. (YATO411), and Pseudomonas sp. (YAET411) using 16S rDNA sequence analysis. The microorganisms were grown at pH 6.871 in a mineral salts medium as described elsewhere (Lin and Li, 2006). DNA from pure cultures was used as the electrophoresis markers in some of the bacterial community studies. For the experiments, 250-ml screw-capped amber glass bottles, each containing 80-ml autoclaved phosphate-buffered mineral salts solution (approx. pH 6.8) autoclaved at 121 1C for 20 min and sealed with Teflon Mininert valves, were shaken continuously in the dark at 150 rpm and 3071 1C. A stainless steel needle fitted to a gas-tight syringe was used to inject MTBE, after which the bottles were re-sealed and shaken for 12 h. This procedure ensured complete dissolution of the MTBE and equilibrated the headspace with the solution. Subsequently, 20 mg l1 mixed-culture inoculum was diluted to 20 ml in deionized water and then introduced to give the same final biomass concentration in each bottle. The inoculum containing the three species noted above, and other organisms, was obtained from a biotrickling filter in a petrochemical wastewater treatment plant treating MTBE and BTEX. MTBE and BTEX solvents (499% pure, Aldrich, Steinheim, Germany) were used as carbon source. Metal ions (Al3+, Zn2+) from concentrated stock solutions of the metals in ionic form (1000 mg l1, Merck), were prepared in distilled water. Substrates (MTBE, BTEX) and metal ions (Al3+ and Zn2+) were added as needed to provide a determining concentration in the bottles. Test cultures and uninoculated control cultures were prepared in triplicate.
2.2. Analytical methods for substrates Because MTBE and BTEX are volatile organic compounds (VOCs), equilibrium between their gas and liquid concentrations is maintained through rapid mass transfer between the gas and aqueous phases. Changes in liquid-phase concentration due to biological reactions are slow when compared to the mass transfer rate between the gas and liquid phase. Therefore, gas-phase measurements closely reflect liquid substrate concentrations for biodegradation experiments. In the biodegradation experiments gas samples were periodically collected from the headspace of each bottle to monitor MTBE degradation using 250-ml gas-tight syringes equipped with Teflon Mininert valve fittings. Samples were then injected onto a gas chromatograph equipped with an RTX-1 capillary column (30 m 0.53 mm) and a flame ionization detector (GC-FID, 14B, Shimadzu, Japan). Helium (99.98% pure) was used as the carrier gas, and nitrogen as a makeup gas. The temperature of the oven was controlled at a constant 105 1C; the injector was at 200 1C; and the detector at 250 1C. MTBE concentrations were quantified against primary standard curves. Metal ion concentrations were determined by using an inductively coupled plasma atomic emission spectrophotometer (ICP–AES, Perkin Elmer, Optima 2000DV, USA).
2.3. DNA extraction DNA was extracted using an improved bead-beating method. A groundwater sample (200 ml) was mixed with 0.8 g 0.106 mm glass beads (Biospec Products, 11079101), 600 ml phenol/chloroform/isoamyl alcohol
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(25:24:1), and 200 ml disruption buffer (50 mM NaCl, 50 mM Tris–HCl pH 8, 5% SDS) in a 1.5-ml screw-cap microcentrifuge tube, which was then filled with the disruption buffer to remove the air. The mixture was homogenized on a Mini Bead Beater (Biospec Products, 3110BX) at 2500 rpm for 2 min. The homogenate was centrifuged at 30,000g for 5 min, and the supernatant was transferred to a fresh tube and extracted with 400-ml chloroform/isoamyl alcohol (24:1). The upper aqueous phase was transferred to a fresh tube and mixed with 240-ml isopropanol. The DNA was precipitated by centrifugation (32,600g, 3 min), and washed with 240ml 70% ethanol. The pellet was air-dried for approx. 1 h at room temperature and then allowed to dissolve in 50-ml TE buffer at 65 1C for 1 h. The concentration of the DNA solution was adjusted to 50 ng ml1, after which the final solution was stored at 4 1C for use as the PCR template.
2.4. Polymerase chain reaction The microbial communities were analyzed by using the PCR–SSCP method as described by Lee et al. (1996) and Schwieger and Tebbe (1998) with modifications. The V3 region of the 16S rDNA, corresponding to the nucleotide positions 334–514 of the Escherichia coli. gene, was amplified with the primers EUB1 (50 -CAGACTCCTACGGGAGGCAGCAG-30 ) and U2 (50 -GTATTACCGCGGCTGCTGGCAC-30 ). The PCR program included an initial denaturation at 94 1C for 5 min, 30 cycles at 94 1C for 30 s, 55 1C for 30 s, and 72 1C for 30 s, followed by a final extension of 72 1C for 5 min. The PCR products of 200 bp were verified by gel electrophoresis on 1.8% agarose gels and stored at 4 1C for further use.
2.5. SSCP gel electrophoresis
2.6. Statistical comparison of SSCP pattern The relative positions of the DNA bands in the SSCP gels were analyzed using LabWork software. Similarities between microbial groups were calculated as Dice indices according to procedures appearing in several reports (Dice, 1945; Eichner et al., 1999; Lapara et al., 2001). Dendrograms were calculated using a clustering algorithm of a UPGMA (unweighted pair group method using arithmetic average) with cluster analysis of similarity indices, constructed by NTSYSpc software (NTSYSpc, version 2.1e, Exeter Software, USA).
3. Results and discussion 3.1. Effects of acclimation periods on microbial community Fig. 1a shows that, after a 30-day acclimation period, different types of substrate resulted in different microbial community structures. Lanes L1, L2, and L3 in Fig. 1a represent communities developed in bi-substrate cultures of MTBE+toluene, MTBE+ethylbenzene and MTBE+benzene, respectively. The total number of bands in these profiles were similar, indicating similar species composition in the different environments. However, the substrate
Fig. 1. (a) SSCP bacterial community profiles from 16S rDNA PCR amplicons during batch tests under mixed substrate conditions for 30 days, and (b) cluster analysis. Box zones in (a) represent similar microbial communities; L1: MTBE+toluene, L2: MTBE+ethylbenzene, L3 : MTBE+benzene, L4: MTBE+m-xylene, L5: MTBE+p-xylene, L6: MTBE+o-xylene.
Substrate remaining (%)
A vertical gel electrophoresis apparatus (Model SE600, Hoefer, San Francisco, USA) was used for SSCP analysis. The electrophoresis was conducted in 10% polyacrylamide gel for 6 h at a constant voltage of 300 V. The gel temperature was maintained at 4 1C using a circulating water bath. The DNA samples were mixed with equal volumes of a denaturing solution (95% formamide, 10 mM NaOH, 0.02% bromophenol blue, 0.02% xylene cyanol, and 20 mM EDTA), heated to 95 1C for 5 min, and snap-frozen on ice before loading. The gels were visualized by using the silver-stain method, sandwiched between two pieces of mylar membrane and dried.
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Fig. 2. Influence of mixed substrate conditions on removal different substrates: (m) benzene; (Open triangle) m-xylene; (’) toluene; (&) pxylene; (K) o-xylene; (J) ethylbenzene.
removal efficiency decreased in the order of MTBE+ toluene4MTBE+ethylbenzene4MTBE+benzene (Fig. 2). The community grown with MTBE+toluene also
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generated the most intense bands, followed by the culture with MTBE+benzene. Removal efficiency is therefore a good indication of substrate quality in supporting dominant microbial species growth, as revealed by changes in band intensity. 3.2. Effects of mixed substrates on microbial community
3.3. Microbial community structure in presence of metal ions The effects of both Al3+ and Zn2+ on MTBE degradation, with metal concentrations of 5 and 10 mg l1, were that in all cases MTBE removal rates were lower in the presence of metal ions in the solution (Fig. 4). At both concentrations, Zn2+ consistently showed greater adverse effects than Al3+. The inhibitory effect was dose-dependent at concentrations from 5 to 10 mg l1, with the effect greater at higher concentration. This result is in agreement with Amor et al. (2001), who found that microorganisms exhibited various growth rate and degradation rate reductions in the presence of different types and concentrations of heavy metals. In the present case, zinc exhibited the highest toxicity. Fig. 5a shows the influence of the metal ions on the SSCP profiles of communities, and Fig. 5b the similarity indices and clustering relationships among these communities. Communities grown in the presence and absence of metal ions exhibit a similarity of 0.65, revealing a small change in community structure due to the ions. This is in stark contrast to the results of co-substrate addition, which generally produces distinctively different community profiles (Fig. 3). Nevertheless, this result agrees with Jacek and Jan (2001) who demonstrated differences in microbial community structure between a heavy metal-contaminated and an uncontaminated site. The concentration appears to have a greater impact on community structure than the type of metal present.
MTBE remaining (%)
Growth in bi-substrate systems of MTBE and o-, m- or p-xylene resulted in similar community structures. Moreover, communities in xylene-containing cultures tended to generate greater band numbers than benzene-containing cultures. Bands from the former also tended to have greater intensity (Fig. 1a, L4–L6). Therefore, communities from xylene-containing cultures can on the basis of their structural similarity be placed in this group (Fig. 1a, L4–L6), and those from benzene-containing cultures in another (Fig. 1a, L1–L3). Degrees of similarity between communities are summarized by a dendrogram constructed using UPGMA (Fig. 1b). Communities from xylene- and benzene-containing cultures again fall into two separate groups with similarity within each group as high as 0.88. The substrate degradation rate has been known to affect community structure (Balcke et al., 2004). Our results show that toluene, ethylbenzene, and benzene are degraded at higher rates than o-, m- and p-xylene (Fig. 2). Aromatic compounds with high degradation rates frequently generate communities with simple structures, probably by favouring the growth of a few dominant species, exemplified in Fig. 3 by the gradual increase in the intensity of
bands a and b in L2/L3 and L8/L9. Such changes demonstrate that the acclimation of communities to a bisubstrate system proceeds over an extended incubation period as indigenous species capable of utilizing the substrates gradually increase in proportion and become the dominant populations. Consistent with these observations, the species represented by bands a and b in the MTBE+toluene culture during the 30-day acclimation period also appeared in pure cultures isolated by using toluene, benzene, or ethylbenzene as the sole carbon source (Fig. 3, L1, L4 and L7, respectively).
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Fig. 3. SSCP bacterial community profiles obtained from 16S rDNA PCR amplicons during batch tests under mixed substrate conditions: L1, YATO411; L2, MTBE+toluene (10 days); L3, MTBE+toluene (30 days); L4, YABE411; L5, MTBE+benzene (10 days); L6, MTBE+benzene (30 days); L7, YAET411; L8, MTBE+ethylbenzene (9 days); L9, MTBE+ethylbenzene (30 days). Arrows denote dominant microbes.
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Fig. 4. Influence of metal ions, at 5 and10 mg l1, on MTBE removal. (W) MTBE alone; (&) MTBE+Zn (5 mg l1); (’) MTBE+Zn(10 mg l1); (J) MTBE+Al(5 mg l1); (K) MTBE+Al(10 mg l1).
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microbial species. The acclimation of communities to a bisubstrate system involves gradual proportional increase of indigenous substrate-utilizing species in relation to their competitors. Consequently, aromatic compounds with higher degradation rates frequently generate communities with simpler structures, probably by favouring the growth of a few dominant species. Here, the presence of metals significantly reduced the diversity of the bacterial community. When the high similarities between SSCP patterns of 16S rDNA fingerprints are judged, it is apparent that greater metal concentration resulted in less growth and differentiation of the microbial community. Acknowledgments
(a) Al (5 mg l-1) Zn (5 mg l-1)
References
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The financial support of the National Science Council of Taiwan, ROC (NSC 93-2211-E-212-007) is gratefully acknowledged. The authors also wish to express appreciation to Dr Cheryl J. Rutledge for her assistance in preparing this manuscript.
0.88
Similarity
Fig. 5. (a) SSCP bacterial community profiles from 16S rDNA PCR amplicons during batch tests for mixed cultures containing MTBE and metals, and (b) cluster analysis: L1, Al (5 mg l1); L2, Zn (5 mg l1); L3, Al (10 mg l1); L4, Zn (10 mg l1); L5, MTBE alone.
Community structures treated with metal ions can be divided into two groups on the basis of ion concentrations. At 5 and 10 mg l1, the similarities between Al- and Zntreated communities were 0.88 and 0.82, respectively. However, the similarity between communities at the two concentrations of Zn or Al was o0.66. Cultures treated with metal ions also contained a smaller number of bands and lower band intensity than the untreated sample. Both these features agree with those of Jacek and Jan (2000), who found that communities from sites contaminated by high concentrations of heavy metals (Pb, 1380 mg kg1; Cd, 23.3 mg kg1; Zn, 2390 mg kg1) were much lower in species diversity than communities from less-contaminated sites (160, 4.4 and 330 mg kg1, respectively). In conclusion, it can be said that, as revealed by band intensity increases in PCR–SSCP community profile analysis, removal efficiency is a good indication of the substrate quality in supporting growth of the dominant
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International Biodeterioration & Biodegradation 59 (2007) 97–102 www.elsevier.com/locate/ibiod
MTBE biodegradation and degrader microbial community dynamics in MTBE, BTEX, and heavy metal-contaminated water Chi-Wen Lina,, Hung-Chun Lina, Chi-Yung Laib a
Department of Environmental Engineering, Da-Yeh University, 112 Shan-Jiau Rd., Da-Tsuen, Changhua 515, Taiwan, ROC b Department of Biology, National Changhua University of Education, Changhua 515, Taiwan, ROC Received 12 March 2006; received in revised form 21 June 2006; accepted 31 July 2006 Available online 26 September 2006
Abstract The aim of this investigation was to explore microbial community changes under various environmental groundwater conditions (single substrate, mixed substrates, and the presence of heavy metals) and link the changes with simultaneously diminishing substrate concentration in the microcosms. Most microorganisms from environmental microcosms or wastewater treatment plants cannot be cultivated artificially. Capturing microbial community fingerprints, therefore, requires applying a molecular biological technique. By using SSCP profiles of PCR-amplified 16S rDNA genes, it was demonstrated that with the repeated addition of substrates during longterm acclimation, substrate-utilizing populations in a microcosm gradually increased to become the dominant constituent. Conversely, the presence of metals inhibited community development and differentiation. It was also shown that substrate degradation rates increased under co-substrate conditions, with substrate-degraders easily adapting to the environment and becoming the dominant bacteria, a phenomenon attributed to the propensity of the fittest species to outgrow their competitors when presented with suitable substrates. r 2006 Elsevier Ltd. All rights reserved. Keywords: 16S rDNA; Metal ions; Microbial community structure; Molecular profiling; Methyl tert-butyl ether
1. Introduction Methyl tertiary butyl ether (MTBE) is one of several fuel oxygenates added to gasoline to replace tetraethyl lead and reduce harmful tailpipe emissions. The present guideline limit established by the US Environmental Protection Agency (EPA) is 20–40 mg l1 for MTBE in drinking water (Jacobs et al., 2001). The EPA also classifies MTBE as a possible human carcinogen (Johnson et al., 2000). Numerous studies have demonstrated that MTBE can be biodegraded under aerobic or anaerobic conditions (Eweis et al., 1997; Mo et al., 1997; Steffan et al., 1997; Hanson et al., 1999; Bruns et al., 2001; Hristova et al., 2003). The MTBE biodegradation rate may further diminish in the presence of co-contaminants, such as benzene, ethylbenzene, toluene, and xylene (BTEX). However, little is known
Corresponding author. Tel.: +886 4 8511339; fax: +886 4 8511347.
E-mail address: [email protected] (C.-W. Lin). 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.08.002
about the microbial community structure during aerobic MTBE degradation in the presence of BTEX. The inhibitory effects on MTBE biodegradation have recently been investigated by several researchers. These studies have focused mainly on substrate inhibition (Park, 1999), by-product inhibition (Liu et al., 2001; Wilson et al., 2002) or competitive inhibition (Garnier et al., 1999; Sedran et al., 2002). However, little has been published on the effect of heavy metals on MTBE biodegradation. Many types of pollutants may coexist in a contaminated site. For instance, organic chemicals and heavy metals can appear simultaneously in a leaking oil tank. Reports by several researchers (Baldrian et al., 2000; Bruins et al., 2000) indicate that some heavy metals (Cu, Zn, Cd) produce toxicity and may also simultaneously affect organic contaminant biodegradation (Said and Lewis, 1991) owing to heavy metal accumulation in the microbial cells. Metal toxicity on microorganisms has been studied by growth inhibition, while heavy metal toxic effects on bacterial community structure have been studied to a
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lesser extent. To fully exploit MTBE bioremediation technology for field application, the biodegradation and microbial community structure in the presence of heavy metals must be better understood. Most microorganisms cannot be detected in soil and groundwater samples using cultivation-based methods (Amann et al., 1995; Ranjard et al., 2000). Conventional microorganism analyses cannot fully reveal the species and the resulting microbial community composition in the environment. Hence, it is necessary to utilize molecular approaches to provide clues to bacterial types involved in aerobic phenol degradation and determine the relationship between growth and decline among bacterial populations. Methods for exploring the microbial community dynamics during in situ bioremediation has recently received increased attention. Griffiths et al. (1999) employed a DNA–phospholipids fatty acid (PLFA) method to analyze the effects of varying substrate concentrations on a soil microbial community. Their findings indicated that, in the substrate range 0–125 mg ml1, lower substrate concentrations resulted in less change in community structure, as evidenced by high similarity indices (0.8–0.9) between preand post-incubation samples. Peter et al. (2000) demonstrated that compost microbial communities are dominated by a few species with the most favoured growth requirements. Hristova et al. (2001) dealt with molecular fingerprinting techniques and detected the MTBE-degrading strain PM1 by real-time TaqMan PCR. Deeb et al. (2001) showed the substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate. These authors have proposed some tools for determining whether certain degraders are present, and have shown inhibition by o-xylene. However, they did not address the changing composition of microbial communities. Zhang et al. (2005) studied a sequencing batch reactor (SBR) system using PCR–denaturing gradient gel electrophoresis (DGGE) and reported that the resident community reached a stable structure dominated by nine species after operation for 200 days. Single-strand-conformation polymorphism (SSCP) is one of the methods frequently used, being inexpensive and highly sensitive (Lee et al., 1996; Schwieger and Tebbe, 1998). In recent years, it has been applied extensively to detect mixed culture diversity in the environment. For instance, Stach et al. (2001) showed that SSCP microbial community analysis could be used to compare the efficiency of alternative methods for DNA extraction and purification. Balcke et al. (2004) used PCR–SSCP to evaluate microbial community changes under consecutive aerobic–anaerobic and various pH conditions. Dassonville et al. (2004) examined microbial dynamics in anaerobic soil slurry and analyzed their dependence on geochemical processes by using the SSCP technique. In this study MTBE biodegradation was analyzed in the presence of other contaminants in batch culture experiments. Some of the contaminants served as co-substrates while the others functioned as growth inhibitors. Then the
SSCP method was combined with batch culture experiments to examine the microbial population and community changes during degradation. 2. Materials and methods 2.1. Bacterial strains and batch microcosm experiments The bacterial cultures used in this study were obtained from a petrochemical wastewater treatment plant. They were initially grown in a liquid medium and adapted to the targeted compounds and working conditions before the biodegradation experiments. After a series of screening and isolation procedures, three pure cultures were identified as Ralstonia sp. (YABE411), Pseudomonas sp. (YATO411), and Pseudomonas sp. (YAET411) using 16S rDNA sequence analysis. The microorganisms were grown at pH 6.871 in a mineral salts medium as described elsewhere (Lin and Li, 2006). DNA from pure cultures was used as the electrophoresis markers in some of the bacterial community studies. For the experiments, 250-ml screw-capped amber glass bottles, each containing 80-ml autoclaved phosphate-buffered mineral salts solution (approx. pH 6.8) autoclaved at 121 1C for 20 min and sealed with Teflon Mininert valves, were shaken continuously in the dark at 150 rpm and 3071 1C. A stainless steel needle fitted to a gas-tight syringe was used to inject MTBE, after which the bottles were re-sealed and shaken for 12 h. This procedure ensured complete dissolution of the MTBE and equilibrated the headspace with the solution. Subsequently, 20 mg l1 mixed-culture inoculum was diluted to 20 ml in deionized water and then introduced to give the same final biomass concentration in each bottle. The inoculum containing the three species noted above, and other organisms, was obtained from a biotrickling filter in a petrochemical wastewater treatment plant treating MTBE and BTEX. MTBE and BTEX solvents (499% pure, Aldrich, Steinheim, Germany) were used as carbon source. Metal ions (Al3+, Zn2+) from concentrated stock solutions of the metals in ionic form (1000 mg l1, Merck), were prepared in distilled water. Substrates (MTBE, BTEX) and metal ions (Al3+ and Zn2+) were added as needed to provide a determining concentration in the bottles. Test cultures and uninoculated control cultures were prepared in triplicate.
2.2. Analytical methods for substrates Because MTBE and BTEX are volatile organic compounds (VOCs), equilibrium between their gas and liquid concentrations is maintained through rapid mass transfer between the gas and aqueous phases. Changes in liquid-phase concentration due to biological reactions are slow when compared to the mass transfer rate between the gas and liquid phase. Therefore, gas-phase measurements closely reflect liquid substrate concentrations for biodegradation experiments. In the biodegradation experiments gas samples were periodically collected from the headspace of each bottle to monitor MTBE degradation using 250-ml gas-tight syringes equipped with Teflon Mininert valve fittings. Samples were then injected onto a gas chromatograph equipped with an RTX-1 capillary column (30 m 0.53 mm) and a flame ionization detector (GC-FID, 14B, Shimadzu, Japan). Helium (99.98% pure) was used as the carrier gas, and nitrogen as a makeup gas. The temperature of the oven was controlled at a constant 105 1C; the injector was at 200 1C; and the detector at 250 1C. MTBE concentrations were quantified against primary standard curves. Metal ion concentrations were determined by using an inductively coupled plasma atomic emission spectrophotometer (ICP–AES, Perkin Elmer, Optima 2000DV, USA).
2.3. DNA extraction DNA was extracted using an improved bead-beating method. A groundwater sample (200 ml) was mixed with 0.8 g 0.106 mm glass beads (Biospec Products, 11079101), 600 ml phenol/chloroform/isoamyl alcohol
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(25:24:1), and 200 ml disruption buffer (50 mM NaCl, 50 mM Tris–HCl pH 8, 5% SDS) in a 1.5-ml screw-cap microcentrifuge tube, which was then filled with the disruption buffer to remove the air. The mixture was homogenized on a Mini Bead Beater (Biospec Products, 3110BX) at 2500 rpm for 2 min. The homogenate was centrifuged at 30,000g for 5 min, and the supernatant was transferred to a fresh tube and extracted with 400-ml chloroform/isoamyl alcohol (24:1). The upper aqueous phase was transferred to a fresh tube and mixed with 240-ml isopropanol. The DNA was precipitated by centrifugation (32,600g, 3 min), and washed with 240ml 70% ethanol. The pellet was air-dried for approx. 1 h at room temperature and then allowed to dissolve in 50-ml TE buffer at 65 1C for 1 h. The concentration of the DNA solution was adjusted to 50 ng ml1, after which the final solution was stored at 4 1C for use as the PCR template.
2.4. Polymerase chain reaction The microbial communities were analyzed by using the PCR–SSCP method as described by Lee et al. (1996) and Schwieger and Tebbe (1998) with modifications. The V3 region of the 16S rDNA, corresponding to the nucleotide positions 334–514 of the Escherichia coli. gene, was amplified with the primers EUB1 (50 -CAGACTCCTACGGGAGGCAGCAG-30 ) and U2 (50 -GTATTACCGCGGCTGCTGGCAC-30 ). The PCR program included an initial denaturation at 94 1C for 5 min, 30 cycles at 94 1C for 30 s, 55 1C for 30 s, and 72 1C for 30 s, followed by a final extension of 72 1C for 5 min. The PCR products of 200 bp were verified by gel electrophoresis on 1.8% agarose gels and stored at 4 1C for further use.
2.5. SSCP gel electrophoresis
2.6. Statistical comparison of SSCP pattern The relative positions of the DNA bands in the SSCP gels were analyzed using LabWork software. Similarities between microbial groups were calculated as Dice indices according to procedures appearing in several reports (Dice, 1945; Eichner et al., 1999; Lapara et al., 2001). Dendrograms were calculated using a clustering algorithm of a UPGMA (unweighted pair group method using arithmetic average) with cluster analysis of similarity indices, constructed by NTSYSpc software (NTSYSpc, version 2.1e, Exeter Software, USA).
3. Results and discussion 3.1. Effects of acclimation periods on microbial community Fig. 1a shows that, after a 30-day acclimation period, different types of substrate resulted in different microbial community structures. Lanes L1, L2, and L3 in Fig. 1a represent communities developed in bi-substrate cultures of MTBE+toluene, MTBE+ethylbenzene and MTBE+benzene, respectively. The total number of bands in these profiles were similar, indicating similar species composition in the different environments. However, the substrate
Fig. 1. (a) SSCP bacterial community profiles from 16S rDNA PCR amplicons during batch tests under mixed substrate conditions for 30 days, and (b) cluster analysis. Box zones in (a) represent similar microbial communities; L1: MTBE+toluene, L2: MTBE+ethylbenzene, L3 : MTBE+benzene, L4: MTBE+m-xylene, L5: MTBE+p-xylene, L6: MTBE+o-xylene.
Substrate remaining (%)
A vertical gel electrophoresis apparatus (Model SE600, Hoefer, San Francisco, USA) was used for SSCP analysis. The electrophoresis was conducted in 10% polyacrylamide gel for 6 h at a constant voltage of 300 V. The gel temperature was maintained at 4 1C using a circulating water bath. The DNA samples were mixed with equal volumes of a denaturing solution (95% formamide, 10 mM NaOH, 0.02% bromophenol blue, 0.02% xylene cyanol, and 20 mM EDTA), heated to 95 1C for 5 min, and snap-frozen on ice before loading. The gels were visualized by using the silver-stain method, sandwiched between two pieces of mylar membrane and dried.
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Fig. 2. Influence of mixed substrate conditions on removal different substrates: (m) benzene; (Open triangle) m-xylene; (’) toluene; (&) pxylene; (K) o-xylene; (J) ethylbenzene.
removal efficiency decreased in the order of MTBE+ toluene4MTBE+ethylbenzene4MTBE+benzene (Fig. 2). The community grown with MTBE+toluene also
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generated the most intense bands, followed by the culture with MTBE+benzene. Removal efficiency is therefore a good indication of substrate quality in supporting dominant microbial species growth, as revealed by changes in band intensity. 3.2. Effects of mixed substrates on microbial community
3.3. Microbial community structure in presence of metal ions The effects of both Al3+ and Zn2+ on MTBE degradation, with metal concentrations of 5 and 10 mg l1, were that in all cases MTBE removal rates were lower in the presence of metal ions in the solution (Fig. 4). At both concentrations, Zn2+ consistently showed greater adverse effects than Al3+. The inhibitory effect was dose-dependent at concentrations from 5 to 10 mg l1, with the effect greater at higher concentration. This result is in agreement with Amor et al. (2001), who found that microorganisms exhibited various growth rate and degradation rate reductions in the presence of different types and concentrations of heavy metals. In the present case, zinc exhibited the highest toxicity. Fig. 5a shows the influence of the metal ions on the SSCP profiles of communities, and Fig. 5b the similarity indices and clustering relationships among these communities. Communities grown in the presence and absence of metal ions exhibit a similarity of 0.65, revealing a small change in community structure due to the ions. This is in stark contrast to the results of co-substrate addition, which generally produces distinctively different community profiles (Fig. 3). Nevertheless, this result agrees with Jacek and Jan (2001) who demonstrated differences in microbial community structure between a heavy metal-contaminated and an uncontaminated site. The concentration appears to have a greater impact on community structure than the type of metal present.
MTBE remaining (%)
Growth in bi-substrate systems of MTBE and o-, m- or p-xylene resulted in similar community structures. Moreover, communities in xylene-containing cultures tended to generate greater band numbers than benzene-containing cultures. Bands from the former also tended to have greater intensity (Fig. 1a, L4–L6). Therefore, communities from xylene-containing cultures can on the basis of their structural similarity be placed in this group (Fig. 1a, L4–L6), and those from benzene-containing cultures in another (Fig. 1a, L1–L3). Degrees of similarity between communities are summarized by a dendrogram constructed using UPGMA (Fig. 1b). Communities from xylene- and benzene-containing cultures again fall into two separate groups with similarity within each group as high as 0.88. The substrate degradation rate has been known to affect community structure (Balcke et al., 2004). Our results show that toluene, ethylbenzene, and benzene are degraded at higher rates than o-, m- and p-xylene (Fig. 2). Aromatic compounds with high degradation rates frequently generate communities with simple structures, probably by favouring the growth of a few dominant species, exemplified in Fig. 3 by the gradual increase in the intensity of
bands a and b in L2/L3 and L8/L9. Such changes demonstrate that the acclimation of communities to a bisubstrate system proceeds over an extended incubation period as indigenous species capable of utilizing the substrates gradually increase in proportion and become the dominant populations. Consistent with these observations, the species represented by bands a and b in the MTBE+toluene culture during the 30-day acclimation period also appeared in pure cultures isolated by using toluene, benzene, or ethylbenzene as the sole carbon source (Fig. 3, L1, L4 and L7, respectively).
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Fig. 3. SSCP bacterial community profiles obtained from 16S rDNA PCR amplicons during batch tests under mixed substrate conditions: L1, YATO411; L2, MTBE+toluene (10 days); L3, MTBE+toluene (30 days); L4, YABE411; L5, MTBE+benzene (10 days); L6, MTBE+benzene (30 days); L7, YAET411; L8, MTBE+ethylbenzene (9 days); L9, MTBE+ethylbenzene (30 days). Arrows denote dominant microbes.
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Fig. 4. Influence of metal ions, at 5 and10 mg l1, on MTBE removal. (W) MTBE alone; (&) MTBE+Zn (5 mg l1); (’) MTBE+Zn(10 mg l1); (J) MTBE+Al(5 mg l1); (K) MTBE+Al(10 mg l1).
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microbial species. The acclimation of communities to a bisubstrate system involves gradual proportional increase of indigenous substrate-utilizing species in relation to their competitors. Consequently, aromatic compounds with higher degradation rates frequently generate communities with simpler structures, probably by favouring the growth of a few dominant species. Here, the presence of metals significantly reduced the diversity of the bacterial community. When the high similarities between SSCP patterns of 16S rDNA fingerprints are judged, it is apparent that greater metal concentration resulted in less growth and differentiation of the microbial community. Acknowledgments
(a) Al (5 mg l-1) Zn (5 mg l-1)
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The financial support of the National Science Council of Taiwan, ROC (NSC 93-2211-E-212-007) is gratefully acknowledged. The authors also wish to express appreciation to Dr Cheryl J. Rutledge for her assistance in preparing this manuscript.
0.88
Similarity
Fig. 5. (a) SSCP bacterial community profiles from 16S rDNA PCR amplicons during batch tests for mixed cultures containing MTBE and metals, and (b) cluster analysis: L1, Al (5 mg l1); L2, Zn (5 mg l1); L3, Al (10 mg l1); L4, Zn (10 mg l1); L5, MTBE alone.
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