Spatial Patterns of Methane Oxidation and Methanotrophic Diversity in Landfill Cover Soils of Southern China
Spatial Patterns of Methane Oxidation and Methanotrophic Diversity in Landfill Cover Soils of Southern China
Journal of Microbiology and Biotechnology. 2015. Apr, 25(4): 423-430
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : August 25, 2014
  • Accepted : October 21, 2014
  • Published : April 28, 2015
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About the Authors
Zi-Fang, Chi
Key Laboratory of Groundwater Resources and Environment, Ministry of Education, Jilin University, Changchun 130021, P.R. China
Wen-Jing, Lu
School of Environment, Tsinghua University, Beijing 100084, P.R. China
Hong-Tao, Wang
School of Environment, Tsinghua University, Beijing 100084, P.R. China

Aerobic CH 4 oxidation is an important CH 4 sink in landfills. To investigate the distribution and community diversity of methanotrophs and link with soil characteristics and operational parameters ( e.g. , concentrations of O 2 , CH 4 ), cover soil samples were collected at different locations and depths from the Mengzi semi-aerobic landfill (SAL) in Yunnan Province of southern China. Specific PCR followed by denaturing gradient gel electrophoresis and realtime PCR were used to examine methanotrophs in the landfill cover soils. The results showed that different locations did harbor distinct methanotroph communities. Methanotrophs were more abundant in areas near the venting pipes because of the higher O 2 concentrations. The depth of 20-25 cm, where the ratio of the CH 4 to O 2 was within the range from 1.3 to 8.6, was more conducive to the growth of CH 4 -oxidizing bacteria. Type II methanotrophs dominated in all samples compared with Type I methanotrophs, as evidenced by the high ratio of Type II to Type I methanotrophic copy numbers (from 1.76 to 11.60). The total copy numbers of methanotrophs detected were similar to other ecosystems, although the CH 4 concentration was much higher in SAL cover soil. Methylobacter and Methylocystis were the most abundant Type I and Type II methanotrophs genera, respectively, in the Mengzi SAL. The results suggested that SALs could provide a special environment with both high concentrations of CH 4 and O 2 for methanotrophs, especially around the vertical venting pipes.
Methane (CH 4 ) is the main cause of the greenhouse effect, being the second largest contributor after carbon dioxide (CO 2 ), with a global warming potential 25 times higher than that of CO 2 . Landfills are ranked as one of the largest sources of anthropogenic CH 4 emissions, constituting 30% and 24% of the anthropogenic CH 4 production in 41 European countries and the USA, respectively, and making up for about 3-7% of global CH 4 emissions [12 , 13 , 23] . Landfills represent point sources of CH 4 to the atmosphere and thus are excellent targets for mitigation [8] . In large landfills, landfill gas can be collected for flaring or power generation, but for older and smaller landfills without gas collection systems, a potent option for the mitigation of emitted CH 4 from landfills is to reduce CH 4 emission in cover soils that can function as a sink for CH 4 [3] .
Aerobic CH 4 oxidation by methanotrophic bacteria belonging to Proteobacteria that use CH 4 as the sole carbon and energy source is one of the most primary CH 4 consumptions in landfill cover soils. About 10% to 100% of the CH 4 generated in landfills is oxidized by methanotrophs [5 , 9] . Traditionally, methanotrophs are divided into two taxonomic groups (Type I and II) based on cell morphology, membrane arrangement, carbon assimilation pathway, and predominant phospholipid fatty acids. Type I methanotrophs include the genera Methylobacter , Methylomicrobium , Methylomonas , Methylocaldum , Methylosphaera , Methylothermus , Methylosarcina , Methylococcus , Methylohalobius , Methyloglobulus , and Methylosoma , and the Type II methanotrophs include Methylocystis , Methylosinus , Methylocella , Methyloferula , and Methylocapsa [14 , 22] . Besides these, anaerobic CH 4 oxidation by some archaea and bacteria has been discovered [24] , but the anaerobic oxidation rate of CH 4 would be at least 1 order of magnitude lower than that of aerobic CH 4 oxidation [11 , 15 , 20] . Environmental factors, including pH [14] , temperature [6 , 26] , NH 4 + -N [18 , 21] , and Cu 2+ [10] , affect the oxidation activity and methanotrophic structure. CH 4 and O 2 are the major key factors for methanotrophs as substrates and thus are very important to the growth and stucture of methanotrophs [4 , 25] .
Semi-aerobic landfills (SALs) with a specially designed leachate collection system could adjust flow-in ambient air, and consequently enhance waste stabilization processes. More than 50% of the area in an SAL is aerated owing to continuous ambient air flow. Thus SAL would be an ideal environment for methanotrophs because of both substrates CH 4 and O 2 being in the same region. However, few studies have conducted on the vertical and horizontal distribution of methanotrophs and relationship with substrate concentrations in SALs. Owing to the rising pressure on mitigating CH 4 emissions from landfills, a better understanding on methanotrophs in landfill covers, especially in semi-aerobic landfills, is much required. Thus, in the present study, the abundance and community structure of methanotrophs from cover soils of the Mengzi SAL, which is the first SAL in China, were investigated using real-time polymerase chain reaction (real time-PCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The objective of the study was to understand the changes in methanotroph communities and abundance in SAL cover soils that may help in designing better management practices for CH 4 emission mitigation in SALs.
Materials and Methods
- Site Study and Sampling
Landfill cover soil samples were collected from the Mengzi SAL, located in Yunnan Province, China. The Mengzi landfill has a landing area of 1.4 × 10 5 m 3 , and was put into service in October 2007, having a refuse treatment capacity of 160 t d -1 . A landfill gas (LFG) collection and utilization system is not carried out and LFG is escaping from the landfill. The cover layer soil is brown clay. Before soil sampling, the component and concentration of landfill gas, including CH 4 , CO 2 , and O 2 , near the sampling site were measured using an infrared gas analyzer (X-am7000; Drager, Germany). Four sites were sampled in the landfill, which are 0.5, 5, 10, and 15 m away from the perforated pipe, as shown in Fig. 1 . Perforated vertical venting pipes are connected to the leachate drainage pipe; ambient air flows through these venting pipes into the waste body. The perforated venting pipes act as outlets of landfill gas produced from the waste body. The locations where sampling was done are listed in Table 1 . Bulk and intact soil core samples were collected and sealed in an ice box, and then transported to the laboratory for further analysis. Basic characteristics of soil samples, including pH, moisture content, organic matter, NH 4 + -N, and Cu 2+ , were measured in accordance with a national standard method of China (GB7830-7892-87) [1] . Each sample was a mix of three parallel samples ( n = 3) with the same distance around the perforated vertical venting pipe.
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Sketch of the semi-aerobic landfill.
Locations and characteristics of samples in the Mengzi landfill.
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aValues are given as the mean ± standard deviation (n = 3).
- DNA Extraction and Real-Time qPCR Analysis
DNA was extracted from semi-aerobic landfill cover soil samples using the FastDNA SPIN Kit for Soil (MP Biotechnology, USA) according to the manufacturer’s instructions. Real-time qPCR of methanotrophs was performed using two primer sets: MB10γ/533R (for Type I), MB9α/533R (for Type II) [16] . The qPCR was conducted using SYBR Premix Ex Taq (Takara, Japan) on an iCycler iQ5 thermocycler (Bio-Rad, USA). The thermal cycler conditions were as follows: an initial stage at 95℃ for 4 min, followed by 41 cycles of 1min at 94℃ and 2 min at 58℃. Data analysis was performed using iCycler software (ver. 1.0.1384.0 CR). Standard curves for Type I and Type II methanotrophs were made as described previously [17] . The detection limits for the two types were 10 2 and 10 3 copies, respectively.
- PCR-DGGE Analysis
The amplification of Type I and Type II methanotrophs was carried out using primers MethT1bR/MethT1dF and MethT2R/27F, respectively. A nested PCR was adopted using the above PCR product as templates, with GC358F (with a 40 bp GC clamp added to primer 358F’s 5’ end) and 517R used as primers. DGGE analysis was conducted using a DCode system (Bio-Rad Laboratories, Hercules, CA, USA). The PCR procedure and DGGE process are described in a previous study [17] . Excised bands from the DGGE gel were purified and sequenced by SinoGenoMax Co., Ltd. (Beijing, China). All nucleotide sequences were aligned using the Clustal X program [2] . Phylogenetic trees were constructed using the neighbor-joining method with MEGA 5.0 software. Hierarchical cluster (heatmap) analysis was performed using the gplots package of R ( ). Reference sequences were obtained from the GenBank database and were included in the phylogenetic trees for comparison. All nucleotide sequences were optimally aligned prior to tree construction. The partial sequences of the 16S rRNA gene of methanotrophs obtained in this study are available from the NCBI database under accession numbers JN166462-JN166474 for Type I methanotrophs and JN166475-JN166492 for Type II methanotrophs.
Results and Discussion
- Properties of Soil Samples and Landfill Gas Components of Cover Layers
Table 1 shows the basic characteristics of the examined cover soils sampled from the Mengzi landfill. Because of the heterogeneity of the cover layer in the landfill, the characteristics of the samples had some differences from each other. The pH values at the four sites were all >7, and sites C and D had higher pH values than sites A and B. The moisture levels at all sampling sites were greater than 24%, suitable for methanotrophs; and moisture levels at the C and D sites were greater than at A and B. The lowest organic matter was seen at site B and the highest at D, but there was no obvious differences between A and C. There was no significant difference in NH 4 + -N levels for sampling sites A, C, and D; site B had a lower content. The Cu 2+ content at the C and D sites was higher than that of A and B.
Fig. 2 shows the results of landfill gas components measured around the perforated vertical pipes of the Mengzi landfill at various depths. Samples closer to the surface and closer to the perforated pipes had more O 2 . In SALs, air is sucked in through the bottom drainage pipe and transported to the refuse through vertical venting pipes, which are connected directly to the bottom drainage pipes. Consequently, the increased O 2 caused lower CH 4 concentrations around the perforated pipes.
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CH4, CO2, and O2 concentrations of samples in the Mengzi landfill.
- Methanotrophic Abundance Based on Real-Time PCR
Ribosomal RNA gene fragments of Type I and Type II methanotrophic bacterial populations were detected in all samples from the Mengzi landfill. One of the landfill samples (A20-25, which generated one of the brightest bands in DGGE) was used to construct the standard curve for Type I and Type II methanotrophs. The obtained R 2 values were greater than 0.99 for both standard curves. Slopes of -3.32 and -3.34 were generated for Type I and II methanotrophs, respectively, which correlated with the efficiency of the PCR (E = 105.0% and 96.8% for Type I and Type II populations, respectively).
Based on the copy numbers from the extracted DNA samples, the 16S rRNA gene copy numbers per gram of soil were calculated using the above general linear model analysis. In general, the quantities of Type I methanotrophs in tested samples of the Mengzi landfill cover layers ranged from 10 5 to 10 6 copies/g dry soil, whereas that of Type II methanotrophs ranged from 10 6 to 10 7 copies/g of soil ( Fig. 3 ). The average methanotroph concentrations in the Mengzi landfill were 1.11 × 10 6 and 6.59 × 10 6 gene copies/g dry soil for Type I and Type II methanotrophs, respectively. The ratio of Type II to Type I methanotrophic copy numbers of these samples at the different sites ranged from 1.76 to 11.60, indicating that Type II methanotrophs account for a significantly higher population of the total methanotrophs in the Mengzi landfill cover layer. The results are in agreement with previous findings that Type II methanotrophs outcompete Type I methanotrophs under similar landfill conditions, where the temperature and moisture provide a more suitable environment (>24%) [6 , 7 , 27] . From redundancy analysis ( Fig. 4 ), we found that all amounts of Methylocaldum , Methylosinus , and Methylomicrobium exhibited a positive correlation with pH, Cu 2+ , MC, OC, and NH 4 + , Methylocystis had a positive correlation with CH 4 levels and a negative correlation with NH 4 + levels, and Methylobacter had a positive correlation with O 2 . Because at site B, the organic matter content and NH 4 + level were significantly lower than other sites, Methylocaldum , Methylomicrobium , and Methylosinus would be less favored, while the abundance of Methylocystis would be higher. We speculate that the abundance of methanotrophs at B site was higher than at other sites because of the predominance of Methylocystis in the landfill cover soil. The higher concentration of Cu 2+ should increase the abundance of Methylosinus (Type II), Methylocaldum (Type I), Methylomicrobium (Type I), and Methylothermus (Type I), while Methylocystis (Type II) and Methylobacter (Type I) levels should be lower. At sites C and D, we found a lower abundance of Type I methanotrophs ( Fig. 3 ), indicating that Methylobacter was the predominant genus of Type I methanotrophs. Type I and Type II methanotrophs were both more abundant closer to the venting pipes where the concentrations of CH 4 and O 2 were higher ( Fig. 3 ). Although other parameters differed among the four sampling sites, we detected no pattern for these differences. Taken together, the results suggest that raising the proportion of O 2 was conducive to the growth and function of microbial action under the high concentration of CH 4 in the SAL.
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Quantification of gene copy numbers of methanotrophs in the Mengzi landfill cover soils.
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Correlation plots of the redundancy analysis (RDA) on the relationship between the environment variables and methanotrophic genus.
Our data are informative about differences in soil depth as well. We found that CH 4 concentration was low in the upper layers, and we found less CH 4 -oxidizing bacteria. The ratio of CH 4 and O 2 of between 1.3 and 8.6 in the middle layers was conducive to the growth of CH 4 -oxidizing bacteria. The average O 2 content was 4.2% in the deeper layers and the number of CH 4 -oxidizing bacteria decreased significantly.
- Methanotrophic Community Structure Based on DGGE
The DGGE profile of the methanotrophic community showed some variations among all samples collected from the Mengzi landfill ( Fig. 5 ). The structure of the Type I methanotrophic community typically varied between the different sampling locations ( Fig. 5 A). In this study, there were two dominant bands, I-2 ( Methylobacter sp.) and I-4 (uncultured bacterium), present in all the samples. The community diversity of Type I methanotrophs changed with location, as indicated by the disappearance of bands I-9 (Gammaproteobacteria bacterium) and I-10 ( Methylocaldum sp.); furthermore, the I-5 ( Methanothermobacter ), I-6 (Gamma proteobacterium), and I-11 ( Methanothermobacter ) bands became the dominant bands in sample B 0-5, which was 5 m away from the perforated pipes and had a low O 2 concentration ( Fig. 2 ) . The community diversity of samples around the perforated pipes changed significantly with distance. The results indicated that the structure of Type I methanotrophs was influenced by O 2 concentration, in accordance with the generally accepted idea that Type I methanotrophs are of O 2 -favoring genera [19] . The structure of the Type II methanotrophic community also changed with different locations, but we did not see a single dominant band in all the samples ( Fig. 5 B). These results also showed that there were more dominant bands near the perforated pipes where the O 2 concentration and moisture content were higher and the CH 4 concentration was lower ( Fig. 2 ). This result indicates that O 2 was a limiting factor for Type II methanotrophs at high CH 4 concentration (>25%), although Type II is generally considered as CH 4 -favoring genera.
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Methanotrophic bacterial communities of the Mengzi landfill revealed by DGGE ((A) Type I; (B) Type II).
In total, 31 bands of the Type I and Type II methanotrophs were excised and sequenced. We found different Type I and Type II methanotrophic populations in the tested samples ( Fig. 6 ). The detected Type I populations were related to several methanotrophic genera, including Methylobacter , Methylothermus , Methylomicrobium , Methylocaldum , and an uncultured bacterium. Methylobacter , Methylothermus , and Methylocaldum were the most prevalent genera in all samples of the Mengzi landfill ( Fig. 6 A). Phylogenetic analysis showed that the most commonly detected Type II methanotroph was Methylocystis . We also detected Type II methanotroph Methylosinus ( Fig. 6 B). Previous studies that used cultivation-dependent and DGGE techniques to investigate the methanotrophic bacterial community structure detected Type I populations (mainly Methylobacter and Methylomicrobium ) and Type II populations (mainly Methylocystis and Methylosinus ) in typical sanitary landfill cover soils [28] . The results indicated that a higher ratio of O 2 to CH 4 would increase more genera of Type I methanotrophs, but Type II methanotrophs would change to homogeneous genera.
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Neighbor-joining tree depicting phylogenetic relationships of methanotrophic bacteria of the Mengzi landfill ((A) Type I; (B) Type II).
The genera-level identification of the methanotroph communities in different samples is shown in Fig. 7 . DGGE detected six main methanotroph genera in all the samples. The relative abundance of the dominant genera of Type I methanotrophs from different samples was determined by DGGE ( Fig. 7 A). The relative abundance of the genera varied greatly. The abundance of Methylobacter decreased from 40.0% to 5.0%, whereas Methylothermus , Methylomicrobium , and Methylocaldum were not detected in A 0-5, B 0-5, and D 0-5 samples. The relative abundance of the dominant genera of Type II methanotrophs was determined by DGGE ( Fig. 7 B). The relative abundance of Methylosinus and Methylocystis varied greatly, and the degree of change was greater than 55%.
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Histograms of relative abundance of methanotrophic genera from different samples in the Mengzi landfill ((A) Type I; (B) Type II). The relative abundance was determined by the corresponding normalized band brightness.
Hierarchically clustered heatmap analysis of the DGGE based on the microbial community profiles at the species level was used to identify the different composition of microbial community structures in different samples ( Fig. 8 ). We found that different locations influenced the community of methanotrophs. A total of 13 species of Type I methanotrophs in different samples were observed, about one sixth of them (2 species) were commonly shared by all samples, and the average numbers of species in all samples were greater than four (>4 species) ( Fig. 8 A). The A 0-5~A 40-45 samples were separated from the B 0-5~D 40-45 samples, suggesting clear distinctions of microbial community structure among them, despite the fact they shared the same source of microbial consortia. A total of 18 species of Type II methanotrophs were observed in the different samples, and the average numbers of species in all samples were greater than three (>3 species) ( Fig. 8 B). B 0-5, C 0-5, and C 20-25 samples were clustered together and were well separated from the other samples, but were distinct from one another. The results indicated that both Type I and Type II methanotrophs were influenced more by CH 4 and O 2 than by other environmental factors under landfill conditions.
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Hierarchical cluster analysis of methanotroph communities in different samples of the Mengzi landfill ((A) Type I; (B) Type II). The y-axis is the clustering of the most abundant OTUs (3% distance) in reads. The OTUs were ordered by species. Sample communities were clustered based on the complete linkage method. The color intensity of the scale indicates the relative abundance of each species. Relative abundance was defined by the corresponding normalized band brightness.
This research project was funded by the National Natural Science Foundation of China (Grant No. 41402206 and 41101225), China Postdoctoral Science Foundation (No. 2013M541311), Youth Science Foundation of Jilin Province (No. 20140520152JH), and Key Laboratory for Solid Waste Management and Environment Safety Open Fund (Grant No. SWMES 2013-07).
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