Vertical profiles of community and activity of methanotrophs in landfill cover soils of different age

Authors


Correspondence

Ruo He, Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China. E-mail: heruo@zju.edu.cn

Abstract

Aim

Aerobic CH4 oxidation is an important process controlling CH4 release from landfills to the atmosphere. The aim of this study was to investigate the link between CH4 oxidation activity and methanotrophs abundance and diversity in landfill cover soils of different age.

Methods and Results

Among the three investigated sites, the highest CH4 emission occurred at the active landfill area with the range of 1371–2242 mg m−2 day. The CH4 oxidation activities of landfill cover soils were 1·07–1·21 μmol g−1 h−1 in the landfill area of 7–16 years, which was 7–17 times higher than those in the active landfill area. The relative abundance of methanotrophs assessed by quantification of pmoA gene was about 1·7 × 106–2·4 × 107 copies g−1 in the landfill cover soils. The CH4 oxidation activity was positively correlated with pmoA copy number in the landfill cover soil of each site, respectively. Type II methanotrophs (Methylocystis) and type I methanotrophs including Methylosoma, Methylocaldum and Methylococcus were all present in the landfill cover soils. Compared to type I methanotroph, type II methanotroph, Methylocystis, was more abundant in the acidic landfill cover soils.

Conclusions

Oxidation activity and community structure of methanotrophs varied with depth and age of landfill cover soils.

Significance and Impact of the Study

These findings provide new fundamental information regarding the activity and diversity of methanotrophs in landfill cover soils of different age that may aid predicting and modelling CH4 flux from landfills.

Introduction

Landfill is a major anthropogenic source of CH4 emission and is estimated to be responsible for 35–69 Tg CH4 per year (Huber-Humer et al. 2008). CH4 emissions from landfills constitute 30 and 24% of the anthropogenic CH4 production in Europe and the US, respectively (IPCC 2007; USEPA 2007; EEA 2008). Aerobic CH4 oxidation is an important process controlling CH4 release from landfills to the atmosphere. It has been reported that 6–96% of emitted CH4 from landfills is oxidized by methanotrophs in landfill cover soils (Barlaz et al. 2004; Börjesson et al. 2004; Einola et al. 2007). In addition to oxidizing CH4 from landfills, landfill cover soils also can function as a sink for atmospheric CH4 (Bogner et al. 1995).

Aerobic methanotroph is one of the primary mediators of CH4 consumption in landfill cover soils (Hanson and Hanson 1996; Semrau et al. 2010). Methanotroph is a unique group of methylotrophic bacteria that utilize CH4 as sole carbon and energy source (Hanson and Hanson 1996). Methanotrophs mainly belong to the Proteobacteria, which can be divided into two taxonomic groups: type I methanotrophs and type II methanotrophs based on their cell morphology, ultra-structure, phylogeny and metabolic pathways. Type I methanotrophs include the genera Methylobacter, Methylomicrobium, Methylomonas, Methylocaldum, Methylosphaera, Methylothermus, Methylosarcina, Methylococcus, Methylohalobius and Methylosoma, which belong to the γ subdivision of the Proteobacteria (Semrau et al. 2010). The type II methanotrophs Methylocystis, Methylosinus, Methylocella and Methylocapsa belong to the α subdivision of the Proteobacteria (Hanson and Hanson 1996; Semrau et al. 2010). Three new isolates belonging to the Verrucomicrobia phylum have been identified as CH4 oxidizers (Dunfield et al. 2007; Pol et al. 2007; Islam et al. 2008). Methylovulum miyakonense with a distinct pmoA was found to belong to type I methanotrophs (Iguchi et al. 2010, 2011). Methylomirabilis oxyfera has been reported to oxidize CH4 bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxidemolecules to dinitrogen and oxygen (Ettwig et al. 2010). In addition to methanotrophs, other microbial guilds have been known to consume CH4, including anaerobic micro-organisms (Nauhaus et al. 2005; Knittel and Boetius 2009).

The first step in aerobic CH4 oxidation to CO2 by aerobic methanotrophs is the conversion of CH4 to methanol catalysed by the enzyme methane monooxygenase (MMO) (Hanson and Hanson 1996). There are two types of MMO: particulate membrane-bound MMO (pMMO) and soluble MMO (sMMO) (Hanson and Hanson 1996; Semrau et al. 2010). pMMO has been reported to be present in all methanotrophs except the genus Methylocella (Dedysh et al. 2000) and Methyloferula (Vorobev et al. 2011). sMMO is present only in some methanotrophs, such as Methylocella palustris (Dedysh et al. 2000) and Methylococcus capsulatus (Nielsen et al. 1997). Functional gene pmoA encoding the key subunit of pMMO has been extensively used as an indicator of the activity and abundance of methanotrophs in environmental samples (Costello and Lidstrom 1999; Bourne et al. 2001; McDonald et al. 2008).

Oxidation activity and community structure of methanotrophs change with environmental factors, including temperature, pH, CH4 and O2 concentrations, NH4+-N and NO3-N contents (Hanson and Hanson 1996; Semrau et al. 2010). The landfill cover soils of different age have different physical and chemical characteristics, such as pH, NH4+-N, NO3-N contents and soil gas composition (Scheutz et al. 2009). However, few studies have been conducted to investigate the link between CH4 oxidation activity and methanotrophs abundance and diversity in landfill cover soils of different age. The objective of this study was to characterize CH4 oxidation in landfill cover soils of different age. Gas composition, CH4 oxidation activity and emission were analysed in landfill cover soils of different age in a subtropical landfill (Dawuao landfill). Quantitative PCR (Q-PCR), terminal restriction fragment length polymorphism (T-RFLP) and cloning were applied to analyse the identity and diversity of methanotrophs in the landfill cover soils.

Materials and methods

Study site and sample collection

Landfill cover soils and gas samples were collected from Dawuao landfill, which is located in Dawuao Mountain in Pingshui Town, Zhejiang Province. The area of Dawuao landfill is about 2·9 × 105 m2, of which the landfilling area is about 1·7 × 105 m2. The landfill is still operational and has received ~2 300 000 ton municipal solid waste (MSW) in the past 21 years. There were no geomembrane and natural impermeable layer beneath the cover soils and no gas venting pipes in Dawuao landfill. Landfill gas (LFG) collection and utilization system is not carried out in the landfill and LFG is escaping from the landfill.

Three sites were sampled in this landfill: A site (29°54′31″N, 120°36′29″E, 90–103 m of elevation), which has been covered by sandy loam for 14–16 years and is entirely covered by vegetation (mainly with grass); B site (29°54′30″N,120°36′31″E, 84–87 m of elevation), near half of which is operational (active use); C site (29°54′36″N,120°36′44″E, 61–66 m of elevation), which has been covered by soil for 7–10 years and is covered by sparse vegetation with degree of coverage under 10% (Table 1). Three subsites were set according to S-type at each sampling site and each subsite was away from another about 10 m. Before sampling soils, the above-ground part of vegetation was removed using a cutter. The landfill cover soils in the B site were collected from the nonworking section. Soil sample was collected by the method described by Bao (2000). Considering the thickness of landfill cover soils (~20 cm), two depth samples (i.e. 0–10 and 10–20 cm depth) were taken from each subsite, and then mixed the same depth soil samples from the three subsites together and subsampled immediately upon return to the laboratory.

Table 1. The physical and chemical properties of landfill cover soils used in this study
Sampling siteGPS positionGranular composition (%)Depth (cm)Water content (%)pHOrganic matter (%)Total nitrogen (g per kg)
>2 mm0·02–2 mm0·002–0·02 mm<0·002 mm
  1. a

    Mean ± standard deviation in the landfill cover soils (n = 3).

A29°54′31″N56·226·29·08·70–1018·1 ± 0·5a4·9 ± 0·42·03 ± 0·070·54 ± 0·11
120°36′29″E
10–2015·8 ± 2·15·5 ± 0·21·41 ± 0·020·43 ± 0·07
B29°54′30″N36·436·614·312·60–109·0 ± 0·15·5 ± 0·10·64 ± 0·020·17 ± 0·09
120°36′31″E10–2014·0 ± 0·35·5 ± 0·31·17 ± 0·040·29 ± 0·13
C29°54′36″N42·233·714·110·00–109·4 ± 1·27·3 ± 0·50·79 ± 0·020·28 ± 0·11
120°36′44″E10–2013·5 ± 0·27·2 ± 0·31·00 ± 0·060·37 ± 0·10

Subsamples for DNA extraction were stored at −20°C. Subsamples for the soil physical and chemical analysis (i.e. soil pH, organic matter, total nitrogen (TN) and particle size) were firstly air-dried and then determined by the methods described by Bao (2000). Soil water content was determined by measuring the loss of soil weight after drying in an oven at 105°C for 16 h to a constant weight.

Gas sampling and analysis

Syringes with 30-cm-length needle were used to collect gas samples at the depth of 5, 10 and 20 cm of landfill cover soils. Syringe was flushed with 30 ml of gas sample prior to collecting ~15 ml of gas sample and injecting 10 ml into N2-flushed glass vials with rubber stoppers, where 10 ml gas was extracted before injecting gas sample. Gas concentrations (i.e. CH4, CO2 and O2) were detected using gas chromatography (GC) equipped with thermal conductivity detector and flame ionization detector (Wang et al. 2011). CH4 oxidation activity was estimated as described by Wang et al. (2011).

CH4 flux

CH4 emission from landfill cover soils was measured by static chamber method. The static chamber with an open base (50 cm long, 50 cm wide and 50 cm high) was made of polymethyl methacrylate. A fan was set in the lid to mix the air inside the chamber. A gas sampling port (~1 cm) fitted with a polymethyl methacrylate tube was installed at the centre of the lid of the chamber. A thermometer was set to record the static chamber temperature. Triplicate 10-ml-gas samples were taken periodically up to 60 min from the static enclosure at 0, 10, 20, 30, 40, 50 and 60 min, respectively, and injected into the gas sampling tubes for CH4 concentration analysis. CH4 fluxes from the landfill cover soils were calculated by eqn (1).

display math(1)

Where F is the CH4 flux from the soil cover, g m−2 per day; 16 is the molar mass of CH4, g mol−1; 273 is the Kelvin of standard temperature (0°C), K; 22·4 is standard volume of 1 mole of an ideal gas at standard temperature and pressure, L mole−1 K−1; V is the static enclosure volume, m3; dc/dt is the change in CH4 concentration over time in the static enclosure, L m−3 day−1; S is the surface area of the static enclosure, m2; T is temperature, K.

DNA extraction and Q-PCR analysis

DNA was extracted from about 0·5 g of soil samples using the E.Z.N.A.™ Soil DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA). Q-PCR of 16S rRNA genes of type I and type II methanotrophs and pmoA gene was performed using the primer sets: U785F (Baker et al. 2003)/MethT1bR (Wise et al. 1999) and U785F/MethT2R (Wise et al. 1999) for 16S rRNA genes of type I and type II methanotroph, respectively; A189F (Holmes et al. 1995)/mb661R (Costello and Lidstrom 1999) for pmoA gene. Q-PCR reaction was conducted in three replicates of 10 μl reactions containing All-in-one qPCR Mix (GeneCopoeia, Inc., Rockville, MD, USA), 4·0 pmol each primer and 1 μl template. Thermal cycler conditions were as follows: an initial stage at 95°C for 10 min; 40 cycles of 95°C for 10 s, 55°C for 30 s and 72°C for 30 s; for 16S rRNA genes of type I and type II methanotrophs. Thermal cycler conditions for the pmoA gene were the same as for 16S rRNA genes of type I and type II methanotrophs except for 58°C of anneal temperature in 40 cycles. Standards were made as described previously (He et al. 2012b). The detection limit by Q-PCR was about 102 copies per reaction for pmoA gene and about 103 copies per reaction for 16S rRNA genes of type I and type II methanotrophs.

PCR amplification and T-RFLP analysis

DNA was investigated using T-RFLP of PCR-amplified pmoA gene. PCRs were carried out using 5′ 6-carboxyfluorescien-labelled A189F and unlabelled mb661R primer with a total reaction volume of 40 μl. The PCR contained final concentrations of 1 × PCR buffer (including MgCl2), 250 μmol l−1 dNTPs, 200 nmol l−1 each primer, 0·5 U Taq DNA polymerase and 1 μl template DNA. Thermal cycler conditions were as follows: denaturation at 94°C for 3 min; 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 40 s; and a final extension step of 72°C for 10 min. PCR products were purified using AxyPrepTMDNA Gel Extraction Kit (Axygen Scientific Inc., Union City, CA, USA) and quantified using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Then 40 ng of PCR product was digested and precipitated as described previously (He et al. 2012a). Pellets were resuspended in 1·0 μl H2O, 0·5 μl of GeneScan™ 500LIZ™ Size Standard (Applied Biosystems, Foster City, CA, USA) and 9·5 μl of deionized formamide, and then run on an ABI 3730XL Genetic Analyzer (Applied Biosystems). T-RFLPs were analysed using GeneMapper software systems (Applied Biosystems). The peak height threshold for T-RFLPs was set at 50 fluorescence units. Terminal restriction fragments (T-RFs) <50 and above 500 base pair (bp) were eliminated from the data sets, and then normalized such that peak heights represent a percentage of the total peak height in each sample.

Cloning and sequence analyses

Total DNA extracted from the six soil samples from the three sites was mixed together and used as template for PCR amplification which was identical thermal cycler conditions and PCR mixture with T-RFLP analysis except for the A189F/mb661R primer set. PCR products were purified using AxyPrepTMDNA Gel Extraction Kit (Axygen Scientific Inc.). Cloning was performed with pMD19-T vector kit according to the manufacturer's instruction (Takara, Dalian, China). Seventy clones were randomly selected for screening plasmid inserts by digestion with restriction endonuclease HaeIII (Fermentas International Inc., Burlington, ON, Canada). DNA fragments were separated by electrophoresis through a 2% agarose gel, and each clone was assigned to an operational taxonomic unit (OTU) that represented a unique restriction fragment length polymorphism (RFLP). One clone from each OTU was sequenced at Beijing Liuhehuada Gene Incorporation (China).

Raw pmoA gene sequences were inspected for chimeras by searching for large sequence regions of unexpected nucleotide changes when compared with the reference sequences. Sequence was aligned with related sequences extracted from GenBank and translated to obtain deduced amino acid sequences. Phylogenetic tree was constructed as described previously (He et al. 2012a). Sequences in this study have been submitted to the GenBank database under accession numbers JX998176-JX998185.

Results

Physical and chemical properties of landfill cover soils

The landfill cover soil in the A site was mainly composed of >2 mm particle, accounting for 56·2%, while >2 mm and 0·02–2 mm particles were the main composition of the landfill cover soils in the B and C sites (Table 1). The landfill cover soils investigated in this study were all classed as gravelly sandy loam. The landfill cover soils in the A and B sites were slightly acidic (pH value of 4·9–5·5), while those in the C site was neutral (pH value of 7·2–7·3). The varied pH of landfill cover soils might be due to the difference of original cover soils, which were taken from different places. Water content, organic matter and TN were all higher in the upper soils (0–10 cm) than those in the deeper landfill cover soils (10–20 cm) in the A site, while higher water content, organic matter and TN were observed in the deeper landfill cover soils in the B and C sites.

Vertical soil gas composition and CH4 emission

Among the investigated three sites, the CH4 concentrations present an increasing trend with depth of landfill cover soil (Table 2). The maximum CH4 concentration was observed at the depth of 20 cm in the B landfill cover soil, reaching 57·9% (v/v) and then decreased to 2·1% (v/v) at the depth of 5 cm. The CH4 concentration at 20 cm depth was much lower (6·5% v/v) in the A site compared to those in the B and C sites. In the three sites, the CH4 concentrations at 10 cm depth were all below 10% (v/v). The CO2 concentration in the landfill cover soils present a similar trend to the CH4 concentration and increased with the depth. The maximum CO2 concentration (15·3% v/v) was observed in the deeper landfill cover of the B site. The CO2 concentrations were both near to zero at the depth of 5 cm in all the three landfill cover soils.

Table 2. Vertical gas composition in landfill cover soils and CH4 emissions
Sampling siteDepth (cm)CH4 concentration (% v/v)CO2 concentration (% v/v)O2 concentration (% v/v)CH4 emission (mg m−2 day)
Mean ± STDaRangebMean ± STDaRangeMean ± STDaRangeMean ± STDcRange
  1. a

    Mean ± standard deviation (n = 30).

  2. b

    Range denotes the range of measured values.

  3. c

    Mean ± standard deviation (n = 9).

A50·0 ± 0·00–0·10·0 ± 0·0 0–016·1 ± 1·413·3–18·0115 ± 1137–233
100·1 ± 0·30–0·90·3 ± 0·7 0–2·614·4 ± 2·210·3–17·5
206·5 ± 6·8 0–27·83·9 ± 3·8 0–8·44·4 ± 2·71·4–7·2
B52·1 ± 1·21·1–4·1 0·0 ± 0·1 0–0·310·4 ± 3·76·1–16·41789 ± 4361371–2242
109·6 ± 1·95·4–11·20·3 ± 0·2 0–0·54·4 ± 2·31·6–9·7
2057·9 ± 2·851·9–61·115·3 ± 2·29·9–17·31·8 ± 0·90·8–4·0
C50·3 ± 0·70–0·30·0 ± 0·0 0–015·2 ± 1·613·6–17·9232 ± 115122–351
101·5 ± 2·20–6·20·6 ± 1·4 0–3·712·9 ± 1·611·0–14·5
2030·4 ± 13·07·4–51·010·4 ± 3·51·8–13·13·4 ± 1·31·2–7·3

The O2 concentration showed a reverse trend to the CH4 and CO2 concentrations in the landfill cover soils. Lower O2 concentration was observed in the deeper landfill cover soil. Compared to the landfill area of 7–16 years (A and C sites), the O2 concentration was lower in the deeper landfill cover of the active landfill area (B site). The O2 concentration increased to 4·4 and 10·4% (v/v) at 10 and 5 cm depth of the B landfill cover soil, which was a little lower than those in the A and C landfill cover soils.

Although near zero CH4 concentration was measured at the depth of 5 cm in the A and C landfill cover soils, the CH4 emission was observed at the two sites, ranging from 7 to 351 mg m−2 per day (Table 2). The highest CH4 emission was detected at the active landfill area (B site), with the range of 1371–2242 mg m−2 per day, which was significantly high than those from the A and C sites. The average CH4 emission from the C site was higher relative to the A site, but there was no significant difference between them.

CH4 oxidation activity

The CH4 oxidation activity was similar at the two depth soils of 0–10 and 10–20 cm in the B and C sites, while it was significantly higher at the depth of 10–20 cm than that at the depth of 0–10 cm in the A site. The highest CH4 oxidation activity was observed in the deeper landfill cover soil of the A site and reached 1·21 μmol g−1 h−1 (Fig. 1). The CH4 oxidation activity was slightly higher at the depth soils of 10–20 cm of the A site and 0–10 cm of the C site compared to others, but there was not significantly different in the CH4 oxidation activity of the landfill cover soils in the A and C sites. In the landfill area of 7–16 years (i.e. A and C sites), the CH4 oxidation activities of landfill cover soils were 1·07–1·21 μmol g−1 h−1, which was 7–17 times higher than those in the active landfill area (B site).

Figure 1.

CH4 oxidation activities of the investigated landfill cover soils. Different letters within the graph refer to significant difference at 5% level based on least significant difference (LSD) method □, 0–10 cm; image_n/jam12263-gra-0001.png, 10–20 cm.

Abundance of methanotrophs

Methanotrophs mainly belong to the Proteobacteria, which can be divided into two taxonomic groups: type I methanotrophs and type II methanotrophs. Q-PCR analysis of pmoA gene showed that the abundance of methanotrophs was about 1·7 × 106–2·4 × 107 copies g−1 in the landfill cover soils (Fig. 2a). The highest abundance of methanotrophs was measured in the deeper landfill cover soil of the B site and reached 2·4 × 107 copies g−1, followed by that in the upper of landfill cover soils of the C sites, and the others were not significantly different at the 5% level.

Figure 2.

Quantitative PCR (Q-PCR) of pmoA gene and 16S rRNA genes of type I and type II methanotrophs in the landfill cover soils. Different letters within the graph refer to significant difference at 5% level based on least significant difference (LSD) method. (a) pmoA gene; (b) 16S rRNA gene of type I methanotrophs; and (c) 16S rRNA gene of type II methanotrophs.

The abundance of type I methanotrophs was 0·4 × 106–1·9 × 107 copies g−1 in the three sites (Fig. 2b). The highest abundance of type I methanotrophs was found in the deeper landfill cover soil of the A site and reached 1·9 × 107 copies g−1, which was similar to that in the deeper landfill cover soil of the B site. In the A and B sites, type I methanotrophs were more abundant in the deeper landfill cover soils compared to the upper landfill cover soils. However, in the C site, the abundance of type I methanotrophs in the upper landfill cover soil was significantly higher than that in deeper landfill cover soil. The lowest abundance of type I methanotrophs was found in the upper landfill cover soil of the B site.

The abundance of type II methanotrophs was 4·0 × 106–8·5 × 107 copies g−1 in the three sites (Fig. 2c). The highest abundance of type II methanotrophs was found in the deeper landfill cover soil of the B site, which was significantly higher than that in the deeper landfill cover soil of the A site. In the C site, the average abundance of type II methanotrophs was higher in the deeper landfill cover soil, but it was not significantly different from that in the upper landfill cover soil. Compared to type I methanotrophs, type II methanotrophs were more abundant in the deeper landfill cover soils of the three sites.

Methanotrophic community structure

To assess the overall methanotrophs community structure in the landfill cover soils, composite DNA sample mixed with 80 ng DNA for each individual sample was used to construct a clone library. Among 70 clones of pmoA gene, 65 clones (93%) showed greatest sequence similarity (96–99%) to Methylocystis sp. KS8 (AJ49035) or Methylocystis sp. M (MSU81596) or Methylocystis sp. M (MSU81596) or Methylocystis sp. 39 (AJ459045) (Fig. 3). Five of the 70 clones (7%) belonged to type I methanotrophs, of which two clones had 97% similarity to Methylococcus capsulatus (AF533666); one clone had 91% similarity to Methylocaldum sp. 05J-I-7 (EU275141); and two clones had 87% sequence similarity to Methylosoma sp. TFB (GQ130273).

Figure 3.

Maximum-likelihood tree of deduced pmoA sequences detected in the landfill cover soils. The tree was constructed with Nitrosomonas europaea (AF037107) as an out-group. The scale bar represents 0·1 substitutions per nucleotide position. Bootstrap values >50 are shown. The number of clones assigned to each operational taxonomic unit (OTU) by restriction fragment length polymorphism (RFLP) is shown in parentheses. Predicted T-RF size (bp) determined by in silico digestion with HhaI is listed.

Predictions of T-RF sizes generated by in silico digestion of the cloning sequence data showed that the 82-bp T-RF was associated with Methylocystis, the 130-bp T-RF was related to Methylosoma and/or Methylocaldum; the 247-bp T-RF was related to Methylococcus (Fig. 4). Type II methanotrophs (Methylocystis) and type I methanotrophs (Methylosoma, Methylocaldum and Methylococcus) were the main methanotrophs in the landfill cover soils, accounting for 92·4–98·9% of the total T-RFs abundance. Type II methanotrophs, Methylocystis dominated in the landfill cover soils, accounted for 42·8–77·1% of the total T-RFs abundance. Among the three sampling sites, the relative abundance of Methylocystis was highest in the B site, accounting for 77·1–78% of the total T-RFs abundance. The relative abundance of Methylocystis in pmoA gene was 67·0–69·3 and 42·8–49·8% in the landfill cover soils of the A and C sites, respectively. Methylosoma and Methylocaldum were also important in the landfill soils. The relative abundance of Methylosoma and/or Methylocaldum was highest in the upper landfill cover soil of the C site, accounting for 48%, followed by that in the deeper landfill cover soil of the C site, and the lowest was observed in the upper landfill cover soil of the B site. Methylococcus was also found to be important in the landfill cover soils. The relative abundance of Methylococcus was highest in the deeper landfill cover soil of the C site, accounting for 20% of the total T-RFs abundance. The relative abundance of Methylococcus was 6·1% in the upper landfill cover soil of the B site and 0·8–2·1% in other landfill cover soils. Compared to the landfill cover soils of the C site, a higher abundance of Methylocystis was observed in the landfill cover soils of the A and B sites.

Figure 4.

Relative abundance of terminal-restriction fragments (T-RFs) of pmoA gene amplicons obtained from the landfill cover soils. The 82-bp T-RF is associated with type II methanotroph Methylocystis, the 130-bp T-RF is related to type I methanotrophs (Methylosoma and/or Methylocaldum); the 247-bp T-RF is related to type I methanotroph Methylococcus. image_n/jam12263-gra-0001.png, 82 bp; image_n/jam12263-gra-0002.png, 130 bp; image_n/jam12263-gra-0003.png, 247 bp; □, Other.

Discussion

In this study, the CH4 emission from the landfill ranged from 7 to 2242 mg m−2 per day (Table 2). The highest CH4 emission was observed at the active landfill area (B site), which was significantly higher than that from the old landfill area of 7–16 years (i.e. A and C sites). This might be mainly attributed to two reasons: (i) CH4 production from landfilled refuse decreased with the age of landfilled refuse, which was present by the lower CH4 concentration in the deeper landfill cover soil in the old landfill area of 7–16 years (i.e. A and C sites) compared to the active landfill area (B site) because of higher stabilization of landfilled refuse (Pohland and Harper 1986); (ii) after exposure to LFG for a long time and/or vegetation growth, the CH4 oxidation activity of landfill cover soils could be increased (Fig. 1), and thus, more CH4 was consumed in the landfill cover soils.

The CH4 oxidation activity was positively correlated with pmoA copy number (the abundance of methanotrophs) in the landfill cover soil of each site, respectively. Similar result was obtained by Henneberger et al. (2011) that oxidation rates and pmoA copy number (the abundance of methanotrophs) were significantly positively correlated in the Lindenstock landfill cover soil. This might be in that CH4 concentration in landfill cover soils was a driving factor in CH4 oxidation, ultimately affecting both methanotrophs abundance (Henneberger et al. 2011). However, there was no linear relationship between CH4 oxidation activities and pmoA copy number when data of all the landfill cover soils of the three sites were analysed together. This might be attributable to that landfill cover soils in each site favoured the growth of different types of methanotrophs, because type I methanotroph is usually low-capacity/high-affinity bacteria, while type II group is a high-capacity/low-affinity methanotroph (Hanson and Hanson 1996). In our previous study, strong linear relationships were observed between the populations of methanotrophic bacteria and CH4 oxidation activity in the waste soil and clay soil, respectively, but no linear relationship was also found between the two parameters when both the experimental soils were considered as a group (He et al. 2007).

Among the three sites, the CH4 oxidation activity was significantly lower in the cover soil of the active landfill area (B site) than others, although the abundance of methanotrophs was highest in the deeper landfill cover soil of the B site (Fig. 2). This was likely owing to that the cover soil of the active landfill area was taken from barren land and was not exposed to CH4-rich environment for a long time, in which methanotrophs probably dormant, existing as cysts or exospores (Bowman et al. 1993). Another reason was that the high CH4 or low oxygen concentration in the landfill cover soils of the B site might inhibit the CH4 oxidation activity. In addition, vegetation in the landfill cover soils of the A and C also could enhance CH4 oxidation activity by increasing oxygen diffusion and excreting exudates (Ding et al. 2005; Bohn et al. 2011). Further studies such as the expression of functional genes of methanotrophs need to be performed to better understand the methanotrophs abundance and their activity.

Both type I and type II methanotrophs were abundant in the landfill cover soils (Fig. 2). Compared to type I methanotrophs, type II methanotrophs were more abundant in the deeper landfill cover soils in the three sites by Q-PCR analysis, demonstrating that the high CH4 and low O2 concentrations in the deeper landfill cover soils favouring the growth type II methanotrophs. Similar results have been reported in rice field soil and in agar diffusion columns that type I methanotrophs grow better in low CH4 and high O2 concentrations whereas type II methanotrophs dominate under high CH4 and low O2 concentrations (Amaral and Knowles 1995; Henckel et al. 2000). In this study, the sum of the abundance of type I and type II methanotrophs was not equal to that of pmoA gene detected by Q-PCR, likely due to that type I and type II methanotrophic primers (U785F/MethT1bR, U785F/Met-hT2R) and pmoA gene primer (A189F/mb661R) could not amplify all known methanotrophs, although they have been widely used to examine methanotroph diversity in environmental samples (McDonald et al. 2008).

Type II methanotrophs (Methylocystis) and type I methanotrophs including Methylosoma, Methylocaldum and Methylococcus were all present in the landfill cover soils. The relative abundance of type I methanotrophs, including Methylosoma, Methylocaldum and Methylococcus, was varied in the landfill cover soil samples, suggesting the composition of type I methanotrophs changed with depth and age of landfill cover soils. Compared with type I methanotrophs, type II methanotrophs Methylocystis species dominated in the landfill cover soils of the A and B sites with the relative abundance of type II methanotrophs reached above 67% (Fig. 4). This might be attributable to the slightly acidic landfill cover soils of the A and B sites, which favoured the growth of the type II methanotrophs Methylocystis. Similar results were also obtained by Wise et al. (1999) and Cébron et al. (2007) that type II methanotrophs, such as Methylocystis and Methylosinus were more abundant in slightly acidic landfill cover soil.

Overall, this study showed that oxidation activity and community structure of methanotrophs varied with the depth and age of landfill cover soils and pH. Type II methanotrophs (Methylocystis) and type I methanotrophs including Methylosoma, Methylocaldum and Methylococcus were abundant in the landfill cover soils. Compared with type I methanotrophs, type II methanotrophs dominated in the acidic landfill cover soils. The composition of type I and type II methanotrophs in the landfill cover soils varied with depth and age of landfill cover soils. Further studies such as the effects of the biotic and abiotic factors on the expression of functional genes of methanotrophs also need to be included to understand the role of landfill cover soil in mitigating CH4 emission from landfills.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China with Grants No. 41001148, No. 51178411 and No. 51108419, Zhejiang Province Natural Science Foundation for Distinguished Young Scholars (LR13E080002) and Fundamental Research Funds for the Central Universities (2012QNA6006).

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