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Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of microbial community structure in landfill leachate
Journal of Hazardous Materials 164 (2009) 1503–1508
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of microbial community structure in landfill leachate Miho Uchida a,∗ , Haruna Hatayoshi b , Aoi Syuku-nobe a , Takefumi Shimoyama b , Toru Nakayama b , Akitsugu Okuwaki a , Tokuzo Nishino b , Hisashi Hemmi b,∗∗ a b
Department of Environmental Chemistry and Ecoengineering, Graduate School of Environmental Studies, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
a r t i c l e
i n f o
Article history: Received 8 February 2008 Received in revised form 14 July 2008 Accepted 19 September 2008 Available online 26 September 2008 Keywords: PCR-DGGE Microbial community Landfill Leachate Hydrogen sulfide
a b s t r a c t The structures of microbial communities in water samples obtained from a landfill site that had been a source of environmental pollution by emitting hydrogen sulfide were elucidated using polymerase chain reaction-denaturing gradient gel electrophoresis. The microbial communities, which consisted of a limited number of major microorganisms, were stable for several months. Microorganisms capable of degrading such chemical compounds as 2-hydroxybenzothiazole and bisphenol A were observed in landfill leachate. Microorganisms responsible for the production of hydrogen sulfide were not the primary microbes detected, even in water samples obtained from the site of gas emission. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Environmental changes resulting from human activities, such as development and the discharge of wastes, significantly affect ecological systems in the environment, including microbial communities. The microorganisms that are well suited to the new environment increase, while those that are unsuited to the environmental conditions decrease. The wide variety of existing microorganisms enables rapid adaptation of the microbial community to a new environment. In recent decades, modern mass-consumption societies have generated huge amounts of waste. A large fraction of this waste has been buried in landfill sites, significantly impacting the environments in the vicinity of the sites [1]. The activity of microorganisms is increased by landfill waste, even in landfill sites for the persistent “five stable
∗ Corresponding author. Present address: Department of Environmental Information Engineering, Tohoku Institute of Technology, Yagiyama-Kasumicho 35-1, Taihaku-ku, Sendai, Miyagi 982-8577, Japan. Tel.: +81 22 305 3925; fax: +81 22 305 3926. ∗∗ Corresponding author. Present address: Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Tel.: +81 52 789 4134; fax: +81 52 789 4120. E-mail addresses: [email protected] (M. Uchida), [email protected] (H. Hemmi). 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.09.074
items”—plastics, gums, metals, glasses and building materials. For example, such “inert type” landfill sites often emit hydrogen sulfide, which can cause serious accidents endangering the health and safety of people [2]. The toxic, putrid gas is thought to arise from sulfate ions, eluted mostly from buried plaster building board waste, due to the activity of sulfate-reducing bacteria. In addition to the occasional elution of heavy metals or chemical compounds from the landfill wastes, the wastes are also expected to have a large impact upon the microbial communities inhabiting the sites [3,4]. Although the microbial communities in such landfill sites have been studied, there is insufficient information for complete understanding of the biological processes that occur in landfill sites [5–7]. The Take-no-uchi inert type landfill site in Miyagi Prefecture, Japan, emitted hydrogen sulfide for several years, resulting in a neighborhood protest movement that culminated in the arrest of executives of the site-managing company on the suspicion of illegal usage of the site. The Miyagi Prefectural Government took measures to correct this environmental problem. Using several vertical gas collection pipes that the company had buried at the site to release gases containing hydrogen sulfide, the Prefectural Government periodically sampled the gases and leachates that oozed from the wastes and accumulated in the pipes (http://www.pref.miyagi.jp/takenouchi). The physical and chemical properties of these samples were analyzed to characterize the landfill wastes. The purpose of the present study was to inves-
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M. Uchida et al. / Journal of Hazardous Materials 164 (2009) 1503–1508
tigate the structures of microbial communities in these water samples over a period of several months using molecular biological techniques, such as polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), to determine the impact of landfills on microbial communities in the environment. In addition, chemical compounds in the samples, which are assumed to arise from the degradation of artificial chemical materials such as gum and plastics buried in the landfill site, were also qualitatively analyzed.
2. Materials and methods 2.1. Sampling All samples were provided by the Waste Management Division of the Department of Environmental and Lifestyle in the Miyagi Prefectural Government. Although the landfill site (Fig. 1) was sampled monthly, the samples taken in every two months between April and December of 2003 were used for analysis. A series of influent (I)
Fig. 1. The outline map of the landfill site. The hatched area is the landfill site. Triangles mean the tops of surrounding mountains. P in a circle means a pump used to exhaust leachate, which has oozed from the site and accumulated in a drainage conduit. The leachate is pumped to the outside area through a pipeline. Closed circles represent the sampling points.
M. Uchida et al. / Journal of Hazardous Materials 164 (2009) 1503–1508
samples were obtained from a small stream that runs from neighboring mountains into the landfill site. The water samples from the gas collection pipe (G) were obtained from pipe No. 8, which served to release hydrogen sulfide-containing gas. The leachate (L) water samples were obtained from the overflow of a pump that removed leachate oozing from the site. All samples were cooled immediately after sampling and were kept cold during transportation. Samples for molecular biological experiments were divided into 50 mL plastic tubes and stored at −20 ◦ C until used. The samples taken for chemical analyses were filtered with 0.2 m membranes, tightly packed into glass bottles, and stored in dark under refrigeration. Immediately after filtration, the total organic carbon (TOC) in each sample was measured using a TOC-5000 total organic carbon analyzer (Shimadzu, Japan). 2.2. PCR-DGGE analysis of microbial community structure One hundred milliliters of each water sample were centrifuged at 9000 × g for 10 min to concentrate the microbial cells. Supernatants were discarded such that the final volume of each solution containing the microbial cells was less than 5 mL. One gram of glass beads was added to the solution, followed by vigorous shaking for 3 min. After centrifugation at 300 × g for 3 min, 700 L of supernatant were transferred to a new tube. Microbial genomes were extracted from the supernatant using a MagExtractor-Genome-Kit (TOYOBO, Japan) according to the manufacturer’s instructions. PCR amplification of 16S/18S rDNA was conducted using the universal primers: 520F, 5 -GTGCCAGCMGCCGCGG-3 ; and, GC1400R, 5 -CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGACGGGCGGTGTGTRC-3 [8]. The extracted microbial genomes served as templates, and ExTaq DNA polymerase (TaKaRa) was used in the reaction. DGGE analyses were performed using the RESOLMAX bipartite slab electrophoresis system (ATTO, Japan). The amplified ca. 900-bp DNA fragments were purified by excising DNA bands from a 0.8% agarose-TAE (40 mM Tris–acetate, pH 7.4, and 1 mM EDTA) gel, followed by concentration using ethanol precipitation. The fragments were then separated using a TAE polyacrylamide (5–8%, w/v) gel with a denaturants gradient ranging in concentration from 15 to 55% [7 M urea and 40% (v/v) formamide as 100%]. The gradient gel was cast using the RAPIDAS slab gel fabrication system (ATTO). Electrophoresis was carried out at a constant voltage of 200 V in TAE buffer at 58 ◦ C. After a 4 h
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electrophoresis, the gel was stained with ethidium bromide and visualized with a UV illuminator. Each of the major DNA bands was excised using a QIAEX II Gel Extract Kit (QIAGEN, Germany) and then cloned using a TOPO TA Cloning Kit (Invitrogen, USA). The sequences of the cloned DNA fragments were analyzed with a BECKMAN COULTER CEQ2000XL DNA sequencer, using T3 and T7 primers. Sequence homology search was performed at the NCBI web site (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using the magablast algorithm. 2.3. Separation and analysis of chemical compounds Each 250 mL sample was loaded onto a column packed with 10 mL XAD-4 hydrophobic resin (Rohm and Haas, USA). The column was washed with 50 mL deionized distilled water and then eluted with 50 mL methanol (5000-times condensed grade). The methanol fraction was concentrated to 2 mL under a stream of nitrogen gas. Before the qualitative analyses, 4 mg/L of 1,1 -biphenyl was added to each concentrated sample as the internal standard. The chemical composition of the concentrated sample was analyzed using a capillary gas chromatography–mass spectrometry (GC–MS). The operating conditions of the GC–MS were: instrument, a HP5973 mass spectrometer equipped with a HP6890 gas chromatography (Hewlett Packard, USA); column, HP-5MS (30 m × 0.25 mm I.D., df = 0.25 m); column temperature, 50 ◦ C for 2 min followed by a 10 ◦ C/min ramp to 250 ◦ C for a 2 min hold; injector temperature, 250 ◦ C; carrier gas (He) flow-rate, 2.0 mL/min; injection mode, split; ion-source temperature, 250 ◦ C; ionizing energy, 70 eV. 3. Results and discussion 3.1. Microbial community analysis of influent water samples The I-series samples, used as the background control, did not consistently yield amplified DNA fragments. The inability to amplify DNA was likely due to the low number of microorganisms in these samples. The electric conductivity values and TOCs of the samples were significantly lower than those of the other samples, i.e., the G and L samples, throughout the entire sampling period, confirming this explanation. Over a one-year period, the TOCs of the I samples ranged from 3.3 to 9.5 mg/L, while those of the G and L samples were 400–1100 and 120–140 mg/L, respectively. In the PCR-DGGE analy-
Fig. 2. PCR-DGGE profiles of leachate samples. (A) I-samples. (B) G-samples. (C) L-samples.
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Table 1 Affiliation of DNA bands from PCR-DGGE analyses. DNA band (accession no.)
Organism with the closest sequence [identified organism with the closest sequence]
Identity (%)
Uncultured bacterium clone HDBW-WB58 (AB237721, isolated from deep subsurface groundwater from sedimentary rock milieu)
98
Uncultured bacterium clone BA021 (AF323760, associated with benzoate degradation in a methanogenic consortium, affiliated in candidate phylum OP9, ref. [8]) [Pelotomaculum thermopropionicum strain SI (AP009389)]
93
G61 (AB373016)
Uncultured bacterium clone BA021 [Moorella thermoacetica strain AMP (AY884087)]
98 87
G81 (AB373017)
Uncultured bacterium clone BA021 [M. thermoacetica strain AMP]
98 87
G82 (AB373018)
Uncultured bacterium clone TPD-2 (AY862519, affiliated in Thermotogae, ref. [9]) [Thermotogales sp. SRI-15 (AF255594)]
98 93
G83 (AB373023)
Uncultured bacterium clone TP4 I (AB330848, isolated from thermophilic anaerobic sludge treating palm oil mill effluent) [Anaerobaculum mobile, type strain NGA (AJ243189)]
99
Uncultured bacterium, isolate cMM319-31 (AJ536814) [Hydrogenophaga taeniospiralis clone SE57 (AY771764)]
99 99
L42 (AB373020)
Uncultured bacterium, isolate cMM319-31 [H. taeniospiralis clone SE57]
99 99
L81 (AB373021)
Uncultured bacterium clone 253ds10 (AY212702) [Acidovorax sp. 98-63833 (AY258065)]
98 98
L82 (AB373022)
Malikia granosa, type strain P1 (AJ627188)
99
Influent water I61 (AB373014) Gas collection pipe water G41 (AB373015)
Leachate L41 (AB373019)
85
91
sis, faint DNA bands, one for each sample, with the same migration rate were observed in the samples taken during June (I6) and October (I10), as shown in Fig. 2A. The sequence of the band in the June sample (I61) was determined to be most closely related to 16S rDNA of an uncultured bacterium isolated from deep subsurface groundwater in a sedimentary rock milieu (Table 1). This result suggests that this bacterium is one of the original major microorganisms in the environment around the landfill site.
the primary microbes in the microbial communities of the landfill site when the samples were collected, because the generation of hydrogen sulfide was decreasing year by year (the concentration of hydrogen sulfide measured two years before the sampling period was ∼10-fold higher). However, the possibility that sulfatereducing bacteria did not inhabit at the sampling locations selected for this study but did inhabit other locations within the landfill site cannot be ruled out.
3.2. Microbial community analysis of gas collection pipe water samples
3.3. Chemical analysis of water samples
In contrast, several amplified DNA bands were observed in each G sample. The PCR-DGGE band patterns of the samples collected between April and December (G4–G12) were very similar, suggesting that the structure of microbial community in the pipe was nearly constant over a half year (Fig. 2B). The bands, G41, G61, and G81, which had the same migration rates, also had nearly identical DNA sequences. DNA sequencing and homology search from databases revealed that the major microorganisms in the samples were most closely related to the following uncultured bacteria: a bacterium associated with benzoate degradation in the methanogenic consortium (included in candidate phylum OP9 [9]); a bacterium associated with anaerobic treatment of phenol in wastewater under thermophilic conditions (included in phylum Thermotogae [10]); and, a bacterium isolated from thermophilicanaerobic-sludge treating palm oil mill effluent (distantly related to Anaelobaculum sp.), respectively (Table 1). The result that sulfate-reducing bacteria were not detected as major microorganisms in the G samples was unexpected because hydrogen sulfide at around 100 ppm was still being emitted and was detected in the gas samples collected from the pipe during the sampling period, according to the source (http://www.pref.miyagi.jp/takenouchi/03-H17 12gasunukikan.pdf). The sulfate-reducing bacteria were no longer
It should be noted that the physical conditions of the gas collection pipe sampled were similar to those of the sites where the 16 S rDNAs of the closely related uncultured microorganisms were isolated. The temperature of the leachate in the pipe was reported to be higher than the ambient temperature throughout the sampling period (in general, 34–38 ◦ C), which is thought to arise from unidentified chemical or microbial reactions such as degradation of chemical compounds proceeding underground at the site. Besides, the water environment at the bottom of the hydrogen sulfide-containing gas-collection pipe is thought to be anaerobic. Because all of the related uncultured microorganisms were detected in the microbial communities associated with the anaerobic and probably thermophilic decomposition of organic compounds, such as benzoate, phenol, and palm oil, it might be possible that the microorganisms in the landfill site were also composing communities with similar microbial activities. Thus, the samples were analyzed for organic compounds. To remove interfering compounds, such as humin, the samples were separated and concentrated with a hydrophobic column chromatography prior to gas chromatography-mass spectrometry analyses. As a result, many chemicals, such as 2-hydroxybenzothiazole, bisphenol A, and N-butylbenzenesulfonamide, were detected in both G and L samples but not in I samples (Fig. 3, Table 2). Most of these chemicals are derived from plastics and sulfurized rubber. These results sug-
M. Uchida et al. / Journal of Hazardous Materials 164 (2009) 1503–1508
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Fig. 3. GC–MS total ion chromatograms of the hydrophobic organic compounds in the water samples. The samples I10, G10, and L10, all sampled in October, were used for the GC–MS analyses of their chemical contents. Asterisks indicate the peaks of the internal standard 1,1 -bisphenyl. Information about numbered compounds is shown in Table 2.
gest that the G water samples could contain microorganisms that participate in the degradation of the aromatic compounds or the formation of them from more complex chemical materials buried in the landfill site, including the major bacteria found in the samples. 3.4. Microbial community analysis of leachate water samples A small number of amplified DNA bands were observed in the PCR-DGGE profile of the L samples collected between April and October (L4–L10). Amplification was not observed in PCR of the December sample for an unknown reason. The bands showed similar migration rates, and the sequence analyses of the bands in the April and August samples indicated that the microbial community inhabiting the sampling point was very stable and that all of the major microorganisms were related to bacteria contained in the family Comamonadaceae, as shown in Fig. 2C (Table 1). It should be noted that this family includes the genus, such as Acidovorax and Commamonas, which are known to play significant roles in biodegradation of aromatic compounds, in denitrification, and in the bioremediation of contaminated environments [11–14]. Thus, it is quite probable that the major microorganisms in the samples are also involved in the biodegradation of the chemical compounds, which were also contained in the L samples (Fig. 3, Table 2).
Table 2 Hydrophobic organic compounds detected by GC–MS from leachate samples. No.a
Organic compound
Major m/z
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
2,5-Dimethyl-2,5-hexanediol 3,5,5-Trimethylhexanoic acid 2-Phenyl-2-propanol Bis(2-ethylhexyl)phthalate Bisphenol A 2,2,6,6-Tetramethyl-4-piperidinol 2,2,6,6-Tetramethyl-4-piperidinone Caprolactam Cyclohexanamine 2-Hydroxybenzothiazole N-Ethyl-4-methylbenzenesulfonamide N-Butylbenzenesulfonamide 1,8-Terpin 1,8-Diaza-2,9-diketocyclotetradecane Unknown
9, 70, 113 57, 83 121 59, 81, 96, 71 219, 228 142, 58 58, 83, 140 55, 113 56 151, 96, 123 91, 155, 184 77, 141, 170 149, 167 112, 55, 86, 226 114, 198
a
The numbers correspond with those used in Fig. 3.
4. Conclusions This study examined the structures of microbial communities in water samples obtained from a stable, final disposal site. Although the concentrations of the chemical compounds detected in the column-concentrated samples, which could be roughly estimated by comparing the intensities of ion peaks of the compounds with that of the internal standard, 1,1-biphenyl, were significantly low, the chemicals seem to have affected the structure of microbial communities in the water environment. Because the numbers of microorganisms in the I samples were very low, the microorganisms in the G and L samples should live and grow in the landfill site, probably by metabolizing synthetic organic materials, such as the aromatic compounds detected in this study, as limited nutrients. In the long-term, such microorganisms would restore the environment surrounding the landfill site by degrading these compounds. Moreover, generation of hydrogen sulfide is also believed to result from adaptation of microbial activity to the altered environment. Detailed information on the microbial characteristics is required for the development of more appropriate methods for the disposal of waste materials without releasing harmful compounds. Acknowledgments This work was partially funded by the Miyagi Prefectural Government, Japan, but does not represent the opinion of the government. The authors are grateful to the staff of the Waste Management Division in the Miyagi Prefectural Government and the Miyagi Prefectural Institute of Public Health and Environment for sampling the landfill leachate. References [1] G. Hamer, Solid waste treatment and disposal: effects on public health and environmental safety, Biotechnol. Adv. 22 (2003) 71–79. [2] S. Lee, Q. Xu, M. Booth, T.G. Townsend, P. Chadik, G. Bitton, Reduced sulfur compounds in gas from construction and demolition debris landfills, Waste Manage. 26 (2006) 526–533. [3] T. Baumann, P. Fruhstorfer, T. Klein, R. Niessner, Colloid and heavy metal transport at landfill sites in direct contact with groundwater, Water Res. 40 (2006) 2776–2786. [4] J.A. Leenheer, J. Hsu, L.B. Barber, Transport and fate of organic wastes in groundwater at the Stringfellow hazardous waste disposal site, southern California, J. Contam. Hydrol. 51 (2001) 163–178. [5] L. Ludvigsen, H. Albrechtsen, D.B. Ringelberg, F. Ekelund, T.H. Christensen, Distribution and composition of microbial populations in a landfill
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[6]
[7]
[8]
[9]
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leachate contaminated aquifer (Grindsted, Denmark), Microb. Ecol. 37 (1999) 197–207. W.F. Roling, B.M. van Breukelen, M. Braster, M.T. Goeltom, J. Groen, H.W. van Verseveld, Analysis of microbial communities in a landfill leachate polluted aquifer using a new method for anaerobic physiological profiling and 16 S rDNA based fingerprinting, Microb. Ecol. 40 (2000) 177–188. W.F. Roling, B.M. van Breukelen, M. Braster, B. Lin, H.W. van Verseveld, Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer, Appl. Environ. Microbiol. 67 (2001) 4619–4629. A. Felske, B. Engelen, U. Nubel, H. Backhaus, Direct ribosome isolation from soil to extract bacterial rRNA for community analysis, Appl. Environ. Microbiol. 62 (1996) 4162–4167. P. Hugenholtzt, T. Huber, Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases, Int. J. Syst. Evol. Microbiol. 53 (2003) 289–293.
[10] H.H. Fang, D.W. Liang, T. Zhang, Y. Liu, Anaerobic treatment of phenol in wastewater under thermophilic condition, Water Res. 40 (2006) 427–434. [11] Y. Hurtubise, D. Barriault, J. Powlowski, M. Sylvestre, Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components, J. Bacteriol. 177 (1995) 6610–6618. [12] M.V. Monferran, J.R. Echenique, D.A. Wunderlin, Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer, Chemosphere 61 (2005) 98–106. [13] A. Hiraishi, S.T. Khan, Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment, Appl. Microbiol. Biotechnol. 61 (2003) 103–109. [14] I.L. Pepper, T.J. Gentry, D.T. Newby, T.M. Roane, K.L. Josephson, The role of cell bioaugmentation and gene bioaugmentation in the remediation of co-contaminated soils, Environ. Health Perspect. 110 (Suppl. 6) (2002) 943–946.
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of microbial community structure in landfill leachate Miho Uchida a,∗ , Haruna Hatayoshi b , Aoi Syuku-nobe a , Takefumi Shimoyama b , Toru Nakayama b , Akitsugu Okuwaki a , Tokuzo Nishino b , Hisashi Hemmi b,∗∗ a b
Department of Environmental Chemistry and Ecoengineering, Graduate School of Environmental Studies, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
a r t i c l e
i n f o
Article history: Received 8 February 2008 Received in revised form 14 July 2008 Accepted 19 September 2008 Available online 26 September 2008 Keywords: PCR-DGGE Microbial community Landfill Leachate Hydrogen sulfide
a b s t r a c t The structures of microbial communities in water samples obtained from a landfill site that had been a source of environmental pollution by emitting hydrogen sulfide were elucidated using polymerase chain reaction-denaturing gradient gel electrophoresis. The microbial communities, which consisted of a limited number of major microorganisms, were stable for several months. Microorganisms capable of degrading such chemical compounds as 2-hydroxybenzothiazole and bisphenol A were observed in landfill leachate. Microorganisms responsible for the production of hydrogen sulfide were not the primary microbes detected, even in water samples obtained from the site of gas emission. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Environmental changes resulting from human activities, such as development and the discharge of wastes, significantly affect ecological systems in the environment, including microbial communities. The microorganisms that are well suited to the new environment increase, while those that are unsuited to the environmental conditions decrease. The wide variety of existing microorganisms enables rapid adaptation of the microbial community to a new environment. In recent decades, modern mass-consumption societies have generated huge amounts of waste. A large fraction of this waste has been buried in landfill sites, significantly impacting the environments in the vicinity of the sites [1]. The activity of microorganisms is increased by landfill waste, even in landfill sites for the persistent “five stable
∗ Corresponding author. Present address: Department of Environmental Information Engineering, Tohoku Institute of Technology, Yagiyama-Kasumicho 35-1, Taihaku-ku, Sendai, Miyagi 982-8577, Japan. Tel.: +81 22 305 3925; fax: +81 22 305 3926. ∗∗ Corresponding author. Present address: Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Tel.: +81 52 789 4134; fax: +81 52 789 4120. E-mail addresses: [email protected] (M. Uchida), [email protected] (H. Hemmi). 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.09.074
items”—plastics, gums, metals, glasses and building materials. For example, such “inert type” landfill sites often emit hydrogen sulfide, which can cause serious accidents endangering the health and safety of people [2]. The toxic, putrid gas is thought to arise from sulfate ions, eluted mostly from buried plaster building board waste, due to the activity of sulfate-reducing bacteria. In addition to the occasional elution of heavy metals or chemical compounds from the landfill wastes, the wastes are also expected to have a large impact upon the microbial communities inhabiting the sites [3,4]. Although the microbial communities in such landfill sites have been studied, there is insufficient information for complete understanding of the biological processes that occur in landfill sites [5–7]. The Take-no-uchi inert type landfill site in Miyagi Prefecture, Japan, emitted hydrogen sulfide for several years, resulting in a neighborhood protest movement that culminated in the arrest of executives of the site-managing company on the suspicion of illegal usage of the site. The Miyagi Prefectural Government took measures to correct this environmental problem. Using several vertical gas collection pipes that the company had buried at the site to release gases containing hydrogen sulfide, the Prefectural Government periodically sampled the gases and leachates that oozed from the wastes and accumulated in the pipes (http://www.pref.miyagi.jp/takenouchi). The physical and chemical properties of these samples were analyzed to characterize the landfill wastes. The purpose of the present study was to inves-
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M. Uchida et al. / Journal of Hazardous Materials 164 (2009) 1503–1508
tigate the structures of microbial communities in these water samples over a period of several months using molecular biological techniques, such as polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), to determine the impact of landfills on microbial communities in the environment. In addition, chemical compounds in the samples, which are assumed to arise from the degradation of artificial chemical materials such as gum and plastics buried in the landfill site, were also qualitatively analyzed.
2. Materials and methods 2.1. Sampling All samples were provided by the Waste Management Division of the Department of Environmental and Lifestyle in the Miyagi Prefectural Government. Although the landfill site (Fig. 1) was sampled monthly, the samples taken in every two months between April and December of 2003 were used for analysis. A series of influent (I)
Fig. 1. The outline map of the landfill site. The hatched area is the landfill site. Triangles mean the tops of surrounding mountains. P in a circle means a pump used to exhaust leachate, which has oozed from the site and accumulated in a drainage conduit. The leachate is pumped to the outside area through a pipeline. Closed circles represent the sampling points.
M. Uchida et al. / Journal of Hazardous Materials 164 (2009) 1503–1508
samples were obtained from a small stream that runs from neighboring mountains into the landfill site. The water samples from the gas collection pipe (G) were obtained from pipe No. 8, which served to release hydrogen sulfide-containing gas. The leachate (L) water samples were obtained from the overflow of a pump that removed leachate oozing from the site. All samples were cooled immediately after sampling and were kept cold during transportation. Samples for molecular biological experiments were divided into 50 mL plastic tubes and stored at −20 ◦ C until used. The samples taken for chemical analyses were filtered with 0.2 m membranes, tightly packed into glass bottles, and stored in dark under refrigeration. Immediately after filtration, the total organic carbon (TOC) in each sample was measured using a TOC-5000 total organic carbon analyzer (Shimadzu, Japan). 2.2. PCR-DGGE analysis of microbial community structure One hundred milliliters of each water sample were centrifuged at 9000 × g for 10 min to concentrate the microbial cells. Supernatants were discarded such that the final volume of each solution containing the microbial cells was less than 5 mL. One gram of glass beads was added to the solution, followed by vigorous shaking for 3 min. After centrifugation at 300 × g for 3 min, 700 L of supernatant were transferred to a new tube. Microbial genomes were extracted from the supernatant using a MagExtractor-Genome-Kit (TOYOBO, Japan) according to the manufacturer’s instructions. PCR amplification of 16S/18S rDNA was conducted using the universal primers: 520F, 5 -GTGCCAGCMGCCGCGG-3 ; and, GC1400R, 5 -CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGACGGGCGGTGTGTRC-3 [8]. The extracted microbial genomes served as templates, and ExTaq DNA polymerase (TaKaRa) was used in the reaction. DGGE analyses were performed using the RESOLMAX bipartite slab electrophoresis system (ATTO, Japan). The amplified ca. 900-bp DNA fragments were purified by excising DNA bands from a 0.8% agarose-TAE (40 mM Tris–acetate, pH 7.4, and 1 mM EDTA) gel, followed by concentration using ethanol precipitation. The fragments were then separated using a TAE polyacrylamide (5–8%, w/v) gel with a denaturants gradient ranging in concentration from 15 to 55% [7 M urea and 40% (v/v) formamide as 100%]. The gradient gel was cast using the RAPIDAS slab gel fabrication system (ATTO). Electrophoresis was carried out at a constant voltage of 200 V in TAE buffer at 58 ◦ C. After a 4 h
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electrophoresis, the gel was stained with ethidium bromide and visualized with a UV illuminator. Each of the major DNA bands was excised using a QIAEX II Gel Extract Kit (QIAGEN, Germany) and then cloned using a TOPO TA Cloning Kit (Invitrogen, USA). The sequences of the cloned DNA fragments were analyzed with a BECKMAN COULTER CEQ2000XL DNA sequencer, using T3 and T7 primers. Sequence homology search was performed at the NCBI web site (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using the magablast algorithm. 2.3. Separation and analysis of chemical compounds Each 250 mL sample was loaded onto a column packed with 10 mL XAD-4 hydrophobic resin (Rohm and Haas, USA). The column was washed with 50 mL deionized distilled water and then eluted with 50 mL methanol (5000-times condensed grade). The methanol fraction was concentrated to 2 mL under a stream of nitrogen gas. Before the qualitative analyses, 4 mg/L of 1,1 -biphenyl was added to each concentrated sample as the internal standard. The chemical composition of the concentrated sample was analyzed using a capillary gas chromatography–mass spectrometry (GC–MS). The operating conditions of the GC–MS were: instrument, a HP5973 mass spectrometer equipped with a HP6890 gas chromatography (Hewlett Packard, USA); column, HP-5MS (30 m × 0.25 mm I.D., df = 0.25 m); column temperature, 50 ◦ C for 2 min followed by a 10 ◦ C/min ramp to 250 ◦ C for a 2 min hold; injector temperature, 250 ◦ C; carrier gas (He) flow-rate, 2.0 mL/min; injection mode, split; ion-source temperature, 250 ◦ C; ionizing energy, 70 eV. 3. Results and discussion 3.1. Microbial community analysis of influent water samples The I-series samples, used as the background control, did not consistently yield amplified DNA fragments. The inability to amplify DNA was likely due to the low number of microorganisms in these samples. The electric conductivity values and TOCs of the samples were significantly lower than those of the other samples, i.e., the G and L samples, throughout the entire sampling period, confirming this explanation. Over a one-year period, the TOCs of the I samples ranged from 3.3 to 9.5 mg/L, while those of the G and L samples were 400–1100 and 120–140 mg/L, respectively. In the PCR-DGGE analy-
Fig. 2. PCR-DGGE profiles of leachate samples. (A) I-samples. (B) G-samples. (C) L-samples.
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Table 1 Affiliation of DNA bands from PCR-DGGE analyses. DNA band (accession no.)
Organism with the closest sequence [identified organism with the closest sequence]
Identity (%)
Uncultured bacterium clone HDBW-WB58 (AB237721, isolated from deep subsurface groundwater from sedimentary rock milieu)
98
Uncultured bacterium clone BA021 (AF323760, associated with benzoate degradation in a methanogenic consortium, affiliated in candidate phylum OP9, ref. [8]) [Pelotomaculum thermopropionicum strain SI (AP009389)]
93
G61 (AB373016)
Uncultured bacterium clone BA021 [Moorella thermoacetica strain AMP (AY884087)]
98 87
G81 (AB373017)
Uncultured bacterium clone BA021 [M. thermoacetica strain AMP]
98 87
G82 (AB373018)
Uncultured bacterium clone TPD-2 (AY862519, affiliated in Thermotogae, ref. [9]) [Thermotogales sp. SRI-15 (AF255594)]
98 93
G83 (AB373023)
Uncultured bacterium clone TP4 I (AB330848, isolated from thermophilic anaerobic sludge treating palm oil mill effluent) [Anaerobaculum mobile, type strain NGA (AJ243189)]
99
Uncultured bacterium, isolate cMM319-31 (AJ536814) [Hydrogenophaga taeniospiralis clone SE57 (AY771764)]
99 99
L42 (AB373020)
Uncultured bacterium, isolate cMM319-31 [H. taeniospiralis clone SE57]
99 99
L81 (AB373021)
Uncultured bacterium clone 253ds10 (AY212702) [Acidovorax sp. 98-63833 (AY258065)]
98 98
L82 (AB373022)
Malikia granosa, type strain P1 (AJ627188)
99
Influent water I61 (AB373014) Gas collection pipe water G41 (AB373015)
Leachate L41 (AB373019)
85
91
sis, faint DNA bands, one for each sample, with the same migration rate were observed in the samples taken during June (I6) and October (I10), as shown in Fig. 2A. The sequence of the band in the June sample (I61) was determined to be most closely related to 16S rDNA of an uncultured bacterium isolated from deep subsurface groundwater in a sedimentary rock milieu (Table 1). This result suggests that this bacterium is one of the original major microorganisms in the environment around the landfill site.
the primary microbes in the microbial communities of the landfill site when the samples were collected, because the generation of hydrogen sulfide was decreasing year by year (the concentration of hydrogen sulfide measured two years before the sampling period was ∼10-fold higher). However, the possibility that sulfatereducing bacteria did not inhabit at the sampling locations selected for this study but did inhabit other locations within the landfill site cannot be ruled out.
3.2. Microbial community analysis of gas collection pipe water samples
3.3. Chemical analysis of water samples
In contrast, several amplified DNA bands were observed in each G sample. The PCR-DGGE band patterns of the samples collected between April and December (G4–G12) were very similar, suggesting that the structure of microbial community in the pipe was nearly constant over a half year (Fig. 2B). The bands, G41, G61, and G81, which had the same migration rates, also had nearly identical DNA sequences. DNA sequencing and homology search from databases revealed that the major microorganisms in the samples were most closely related to the following uncultured bacteria: a bacterium associated with benzoate degradation in the methanogenic consortium (included in candidate phylum OP9 [9]); a bacterium associated with anaerobic treatment of phenol in wastewater under thermophilic conditions (included in phylum Thermotogae [10]); and, a bacterium isolated from thermophilicanaerobic-sludge treating palm oil mill effluent (distantly related to Anaelobaculum sp.), respectively (Table 1). The result that sulfate-reducing bacteria were not detected as major microorganisms in the G samples was unexpected because hydrogen sulfide at around 100 ppm was still being emitted and was detected in the gas samples collected from the pipe during the sampling period, according to the source (http://www.pref.miyagi.jp/takenouchi/03-H17 12gasunukikan.pdf). The sulfate-reducing bacteria were no longer
It should be noted that the physical conditions of the gas collection pipe sampled were similar to those of the sites where the 16 S rDNAs of the closely related uncultured microorganisms were isolated. The temperature of the leachate in the pipe was reported to be higher than the ambient temperature throughout the sampling period (in general, 34–38 ◦ C), which is thought to arise from unidentified chemical or microbial reactions such as degradation of chemical compounds proceeding underground at the site. Besides, the water environment at the bottom of the hydrogen sulfide-containing gas-collection pipe is thought to be anaerobic. Because all of the related uncultured microorganisms were detected in the microbial communities associated with the anaerobic and probably thermophilic decomposition of organic compounds, such as benzoate, phenol, and palm oil, it might be possible that the microorganisms in the landfill site were also composing communities with similar microbial activities. Thus, the samples were analyzed for organic compounds. To remove interfering compounds, such as humin, the samples were separated and concentrated with a hydrophobic column chromatography prior to gas chromatography-mass spectrometry analyses. As a result, many chemicals, such as 2-hydroxybenzothiazole, bisphenol A, and N-butylbenzenesulfonamide, were detected in both G and L samples but not in I samples (Fig. 3, Table 2). Most of these chemicals are derived from plastics and sulfurized rubber. These results sug-
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Fig. 3. GC–MS total ion chromatograms of the hydrophobic organic compounds in the water samples. The samples I10, G10, and L10, all sampled in October, were used for the GC–MS analyses of their chemical contents. Asterisks indicate the peaks of the internal standard 1,1 -bisphenyl. Information about numbered compounds is shown in Table 2.
gest that the G water samples could contain microorganisms that participate in the degradation of the aromatic compounds or the formation of them from more complex chemical materials buried in the landfill site, including the major bacteria found in the samples. 3.4. Microbial community analysis of leachate water samples A small number of amplified DNA bands were observed in the PCR-DGGE profile of the L samples collected between April and October (L4–L10). Amplification was not observed in PCR of the December sample for an unknown reason. The bands showed similar migration rates, and the sequence analyses of the bands in the April and August samples indicated that the microbial community inhabiting the sampling point was very stable and that all of the major microorganisms were related to bacteria contained in the family Comamonadaceae, as shown in Fig. 2C (Table 1). It should be noted that this family includes the genus, such as Acidovorax and Commamonas, which are known to play significant roles in biodegradation of aromatic compounds, in denitrification, and in the bioremediation of contaminated environments [11–14]. Thus, it is quite probable that the major microorganisms in the samples are also involved in the biodegradation of the chemical compounds, which were also contained in the L samples (Fig. 3, Table 2).
Table 2 Hydrophobic organic compounds detected by GC–MS from leachate samples. No.a
Organic compound
Major m/z
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
2,5-Dimethyl-2,5-hexanediol 3,5,5-Trimethylhexanoic acid 2-Phenyl-2-propanol Bis(2-ethylhexyl)phthalate Bisphenol A 2,2,6,6-Tetramethyl-4-piperidinol 2,2,6,6-Tetramethyl-4-piperidinone Caprolactam Cyclohexanamine 2-Hydroxybenzothiazole N-Ethyl-4-methylbenzenesulfonamide N-Butylbenzenesulfonamide 1,8-Terpin 1,8-Diaza-2,9-diketocyclotetradecane Unknown
9, 70, 113 57, 83 121 59, 81, 96, 71 219, 228 142, 58 58, 83, 140 55, 113 56 151, 96, 123 91, 155, 184 77, 141, 170 149, 167 112, 55, 86, 226 114, 198
a
The numbers correspond with those used in Fig. 3.
4. Conclusions This study examined the structures of microbial communities in water samples obtained from a stable, final disposal site. Although the concentrations of the chemical compounds detected in the column-concentrated samples, which could be roughly estimated by comparing the intensities of ion peaks of the compounds with that of the internal standard, 1,1-biphenyl, were significantly low, the chemicals seem to have affected the structure of microbial communities in the water environment. Because the numbers of microorganisms in the I samples were very low, the microorganisms in the G and L samples should live and grow in the landfill site, probably by metabolizing synthetic organic materials, such as the aromatic compounds detected in this study, as limited nutrients. In the long-term, such microorganisms would restore the environment surrounding the landfill site by degrading these compounds. Moreover, generation of hydrogen sulfide is also believed to result from adaptation of microbial activity to the altered environment. Detailed information on the microbial characteristics is required for the development of more appropriate methods for the disposal of waste materials without releasing harmful compounds. Acknowledgments This work was partially funded by the Miyagi Prefectural Government, Japan, but does not represent the opinion of the government. The authors are grateful to the staff of the Waste Management Division in the Miyagi Prefectural Government and the Miyagi Prefectural Institute of Public Health and Environment for sampling the landfill leachate. References [1] G. Hamer, Solid waste treatment and disposal: effects on public health and environmental safety, Biotechnol. Adv. 22 (2003) 71–79. [2] S. Lee, Q. Xu, M. Booth, T.G. Townsend, P. Chadik, G. Bitton, Reduced sulfur compounds in gas from construction and demolition debris landfills, Waste Manage. 26 (2006) 526–533. [3] T. Baumann, P. Fruhstorfer, T. Klein, R. Niessner, Colloid and heavy metal transport at landfill sites in direct contact with groundwater, Water Res. 40 (2006) 2776–2786. [4] J.A. Leenheer, J. Hsu, L.B. Barber, Transport and fate of organic wastes in groundwater at the Stringfellow hazardous waste disposal site, southern California, J. Contam. Hydrol. 51 (2001) 163–178. [5] L. Ludvigsen, H. Albrechtsen, D.B. Ringelberg, F. Ekelund, T.H. Christensen, Distribution and composition of microbial populations in a landfill
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[6]
[7]
[8]
[9]
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leachate contaminated aquifer (Grindsted, Denmark), Microb. Ecol. 37 (1999) 197–207. W.F. Roling, B.M. van Breukelen, M. Braster, M.T. Goeltom, J. Groen, H.W. van Verseveld, Analysis of microbial communities in a landfill leachate polluted aquifer using a new method for anaerobic physiological profiling and 16 S rDNA based fingerprinting, Microb. Ecol. 40 (2000) 177–188. W.F. Roling, B.M. van Breukelen, M. Braster, B. Lin, H.W. van Verseveld, Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer, Appl. Environ. Microbiol. 67 (2001) 4619–4629. A. Felske, B. Engelen, U. Nubel, H. Backhaus, Direct ribosome isolation from soil to extract bacterial rRNA for community analysis, Appl. Environ. Microbiol. 62 (1996) 4162–4167. P. Hugenholtzt, T. Huber, Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases, Int. J. Syst. Evol. Microbiol. 53 (2003) 289–293.
[10] H.H. Fang, D.W. Liang, T. Zhang, Y. Liu, Anaerobic treatment of phenol in wastewater under thermophilic condition, Water Res. 40 (2006) 427–434. [11] Y. Hurtubise, D. Barriault, J. Powlowski, M. Sylvestre, Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components, J. Bacteriol. 177 (1995) 6610–6618. [12] M.V. Monferran, J.R. Echenique, D.A. Wunderlin, Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer, Chemosphere 61 (2005) 98–106. [13] A. Hiraishi, S.T. Khan, Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment, Appl. Microbiol. Biotechnol. 61 (2003) 103–109. [14] I.L. Pepper, T.J. Gentry, D.T. Newby, T.M. Roane, K.L. Josephson, The role of cell bioaugmentation and gene bioaugmentation in the remediation of co-contaminated soils, Environ. Health Perspect. 110 (Suppl. 6) (2002) 943–946.