Fig. 1 Terminal restriction fragments (HaeIII digest) and their abundances in the Samples A and B
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College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
1.College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
纸质出版日期: 2011-09 ,
收稿日期: 2010-12-20 ,
修回日期: 2011-08-18 ,
录用日期: 2011-03-08
引用本文
Shu-ying Zhang, Qing-feng Wang, Rui Wan, 等. Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil[J]. 浙江大学学报(英文版)(B辑:生物医学和生物技术), 2011, 12(9):760-768.
Shu-ying Zhang, Qing-feng Wang, Rui Wan, et al. Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2011, 12(9):760-768.
Shu-ying Zhang, Qing-feng Wang, Rui Wan, 等. Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil[J]. 浙江大学学报(英文版)(B辑:生物医学和生物技术), 2011, 12(9):760-768. DOI: 10.1631/jzus.B1000440.
Shu-ying Zhang, Qing-feng Wang, Rui Wan, et al. Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2011, 12(9):760-768. DOI: 10.1631/jzus.B1000440.
College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in a municipal solid waste (MSW) composting site. Knowledge of changes in microbial structure is useful to identify particular PAH degraders. However, the microbial community in the MSW composting soil and its change associated with prolonged exposure to PAHs and subsequent biodegradation remain largely unknown. In this study, anthracene was selected as a model compound. The bacterial community structure was investigated using terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene clone library analysis. The two bimolecular tools revealed a large shift of bacterial community structure after anthracene amendment and subsequent biodegradation. Genera Methylophilus, Mesorhizobium, and Terrimonas had potential links to anthracene biodegradation, suggesting a consortium playing an active role.
With rapid urbanization, municipal solid waste (MSW) constitutes an immediate and serious environmental problem in many developing countries. In many Chinese urban and suburban regions, a great amount of collected MSW is dumped on land in a more or less uncontrolled manner. Leachate from MSW composting site may contain a large number of xenobiotic organic compounds (Baun et al.,
Microbial degradation plays the primary role in PAH reduction in contaminated sites (Yuan et al.,
Anthracene has been listed as one of the priority environmental pollutants by the United States Environmental Protection Agency. Anthracene is also a model compound for PAH degradation studies (Müncnerová and Augustin,
Soil was collected from the vicinity of an unmanaged MSW composting site in Changzhou, a southeast city in China. Following sample collection, soil was air-dried, homogenized, and sieved through a 0.18-mm screen, and stored at 4 °C until use. Anthracene (99%, J & K China Chemical) dissolved in acetone was added to each empty microcosm chamber (150 ml serum bottle) with a total mass of 200 µg anthracene per chamber. After the acetone evaporated, 2 g dry soil was added to each microcosm along with 10 ml phosphate buffered mineral media, as previously described (Mu and Scow,
In order to determine anthracene in solid phase, the water/soil mixture in microcosms was transferred to a 100-ml centrifuge tube. After 10-min centrifugation at 5 000 r/min, the supernatant was collected. A total of 1 g dry soils were extracted three times with 10 ml acetone, using a 300-W ultrasonic processor. The mixture was vigorously shaken and then centrifuged at 5 000 r/min for 10 min. Then 0.2 ml of the supernatant was collected into a gas chromatography (GC) vial and diluted by 0.8 ml methanol (Zhang et al.,
Soil DNAs from Samples A (from microcosm on Day 0) and B (from microcosm on Day 15) were used for further molecular analysis. DNA was extracted using the UltraClean DNA extraction kit (Mo Bio Laboratories). Bacterial 16S rRNA genes were amplified using the bacterial primers 27F-FAM (5′-GAG TTT GAT CMT GGC TCA G-3′, 5′ end-labeled with carboxyfluorescein) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). The polymerase chain reaction (PCR) program was as previously described (Cupples and Sims,
Replicate profiles from separate DNA extractions and PCR reactions for each sample were compared to identify the subset of reproducible fragment sizes. The average area of each reproducible peak was calculated. Terminal fragments smaller than 50 bp or under 200 fluorescent units were excluded from the further analysis. Fragments differing by less than 1 bp length were clustered. The standardized binning criteria used to identify the subset of reproducible peaks were as previously described (Dunbar et al.,
The PCR conditions were the same as the above-mentioned, except that the forward primer was unlabeled. The PCR products were cloned into pMD19-T vector (TaKaRa Co., Japan) following the manufacturer’s instruction. The white colonies were verified by PCR with primers M13 F (5′-TGT AAA ACG ACG GCC AGT-3′) and M13 R (5′-AAC AGC TAT GAC CAT G-3′). Clones containing an insert of the correct size were bidirectionally sequenced. The 69–72 clones recovered from each sample were successfully sequenced in this study. The partial 16S rRNA gene sequences were deposited with GenBank under accession Nos. HQ015161-HQ015301.
Sequences for chimerism were checked using the Chimera Check program available at the Michigan State University (MSU) Center for Microbial Ecology. All clones displaying chimeric profiles were discarded from the further analysis. Sequences that were over 97% similar were grouped into an operational taxonomic unit (OTU) by manual comparison. The representative GenBank sequences to the clones of interest were extracted from the National Center for Biotechnology Information (NCBI) database and included in further phylogenetic analyses using MEGA Version 4.0 (Tamura et al.,
In this study, the total amount of anthracene in supernatant was below 3% of that in the solid phase over the whole experiment (data not shown). Anthracene concentration in soil declined greatly after a 15-d experiment period. In contrast, only a limited decline in soil was observed in the autoclaved control, possibly due to aging effect. This confirmed a biological removal mechanism in microcosms (Table
Time (d) | Remaining ANT (%) | |
---|---|---|
Sterile control with ANT | Microcosm with ANT | |
0 | 97.5 | 97.2 |
10 | 80.9 | 44.8 |
15 | 77.0 | 11.4 |
The total average numbers of terminal restriction fragments (HaeIII digest) in Samples A and B were 35 and 29, respectively (Fig.
Fig. 1 Terminal restriction fragments (HaeIII digest) and their abundances in the Samples A and B
Samples | S | H | E |
---|---|---|---|
A | 35 | 3.191 | 0.897 |
B | 29 | 3.145 | 0.934 |
S: ribotype richness; S=total number of bands in profile.H: Shannon diversity index; H=−∑(pi)(log2pi), where pi is the individual peak area; Hmax=log2S. E: evenness; E=H/Hmax
The SIMPER analysis revealed that the differences of TRFLP profiles (HaeIII digest) between Samples A and B were driven primarily by variation in 217, 79, 222, and 258 bp (Table
Fragment (bp) | Mean relative abundance (%) | Fragment contribution (%) | |
---|---|---|---|
Sample A | Sample B | ||
217 | 0 | 10 | 6 |
79 | 10 | 0 | 6 |
222 | 0 | 8 | 5 |
258 | 0 | 8 | 5 |
Mean relative abundance of each fragment (HaeIII digest) as a percentage of total fragment abundance
Fragment contribution as a dissimilarity percentage between the two groups
Lists are truncated to include only those fragments that contribute no less than 5% to the differences between samples
In this study, many known phyla (Bacteroidetes, Proteobacteria, Verrucomicrobia, Acidobacteria, TM7, Cyanobacteria, Gemmatimonadetes) were detected, although only Bacteroidetes and Proteobacteria were shared between both samples (Fig.
Fig. 2 Percentages of the clones affiliated with different phyla and sub-phyla to the total number of clones from Sample A or B
Clones not classified to any known phylum are included as unclassified bacteria
Moreover, the 72 clones in the bacterial library constructed with Sample A could be further divided into 46 OTUs (14 OTUs had two or more clones). However, the 69 clones in library constructed with Sample B were grouped into 39 OTUs (11 OTUs had two or more clones). Therefore, in agreement with the TRFLP analysis, the bacterial community structure reflected by the clone library analysis also indicated a big change with PAH biodegradation.
The taxonomic identity of 217, 222, or 258 bp (HaeIII digest) was also investigated. Comparisons of TRFLP fragments and in silico cut sites are presented in Table
Enriched fragment | Restriction enzyme | TRFLP | Sequence data |
---|---|---|---|
1 | HaeIII | 217 | 219 |
MspI | 487 | 490 | |
HhaI | 367 | 367 | |
2 | HaeIII | 222 | 225 |
MspI | 128 | 128 | |
HhaI | 58 | 61 | |
3 | HaeIII | 258 | 260 |
MspI | 90 | 93 | |
HhaI | 69 | 67 |
Fragment (bp) | Phylum | Class | Order | Family | Genus |
---|---|---|---|---|---|
217 | Proteobacteria | β-proteobacteria | Methylophilales | Methylophilaceae | Methylophilus |
222 | Proteobacteria | α-proteobacteria | Rhizobiales | Phyllobacteriaceae | Mesorhizobium |
258 | Bacteroidetes | Sphingobacteria | Sphingobacteriales | Chitinophagaceae | Terrimonas |
Phylogenetic tree of two representative sequences in each genus (Methylophilus, Mesorhizobium, or Terrimonas) was constructed using MEGA Version 4.0 (Fig.
Fig. 3 Phylogenetic tree of representative bacterial 16S rRNA gene sequences (beginning with ‘L’) within the genera of Methylophilus, Mesorhizobium, and Terrimonas from Sample B and reference sequences from GenBank
Data in parentheses are GenBank accession numbers. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analysis of 1 000 resampled datasets
Considerable attention has been paid to the diversity of indigenous microorganisms capable of degrading pollutants in various environments (Abed et al.,
The bacterial community structure usually changes during the bioremediation of PAH-contaminated soils (MacNaughton et al.,
The phylogenetic description of the bacterial community structure is also important. Tian et al. (
Many different bacterial genera previously isolated from PAH-contaminated sites belong to Sphingomonas (Pinyakong et al.,
Some species of Methylophilus have been linked to degradation or utilization of various compounds including dichloromethane (Nikolausz et al.,
Mesorhizobium species are usually isolated from root nodules (Lin et al.,
Both TRFLP and 16S rRNA gene clone library analysis revealed the big shift of bacterial community structure in MSW composting soil after anthracene amendment and subsequent biodegradation. Three genera, Methylophilus, Mesorhizobium, and Terrimonas, had potential links to anthracene biodegradation, suggesting a consortium playing an active role. Further study will be necessary to explore the biodegradation mechanism of anthracene in MSW composting soil. This is very important because the presence of particular microorganisms or a consortium may determine the bioremediation strategies for PAHs in MSW composting soil in practice.
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