Introduction

Rice fields are considered as one of the most important sources of atmospheric methane. The aerobic methanotrophs, as a biofilter of methane into atmosphere, are crucial for the attenuation of methane emission from rice fields. At present, all known aerobic methanotrophs belong to two bacterial phyla, the Proteobacteria and the Verrucomicrobia. The verrucomicrobial methanotrophs are restricted to geothermal environments and therefore are not relevant to rice fields [1, 2]. In contrast, the proteobacterial methanotrophs have been proven to play major roles in methane oxidation in paddy soils, and their community structures, abundances and activities have been studied extensively [3–6].

The proteobacterial methanotrophs are generally classified into two groups, type I (belonging to Gammaproteobacteria) and type II (belonging to Alphaproteobacteria) [7, 8]. Type II methanotrophs, especially Methylocystis-like species, are often found to be more abundant than type I methanotrophs in paddy soils [4, 9–11]. In contrast, accumulating evidences showed that the active methanotrophs in paddy soils are type I groups rather than type II [6, 12–14]. So, the ecophysiology of type II methanotrophs in paddy soil remains largely unknown.

For a long time, it was believed that all methanotrophs were obligately methanotrophic, meaning they were able to metabolize methane but not multi-carbon substrates as their sole carbon and energy source. However, the last decade has seen the description of several facultative methanotrophs [15]. Species of the genus Methylocella isolated from acidic Sphagnum peat were the first to be recognized as able to use a variety of multi-carbon compounds, including acetate [16]. Recently, Methylocapsa aurea KYGT from forest soil and some species of the genus Methylocystis from peat or bog samples were reported to have the capacity for facultative growth on acetate [17–19]. In addition, a labelling study showed that the atmospheric methane oxidizers (USC alpha clade) in forest soil assimilate 13C-acetate [20]. Altogether, these findings showed that facultative methanotrophs are more diverse than previously thought, although all currently known facultative species belong to the Alphaproteobacteria methanotrophs. Additionally, facultative methanotrophs might be widespread in many natural environments [21].

Acetate is the most important intermediate in the anaerobic decomposition of organic matter in paddy soil. Its concentration can reach several millimolar or even higher, especially when rice straw or root is anaerobically degraded [3, 22]. Therefore, we hypothesize that the dominant type II methanotrophs in paddy soil might have the facultative capacity to use acetate for growth. In the present study, the methanotrophs in a Chinese paddy soil were enriched by supplying methane and/or acetate. The structure and abundance of methanotrophic community were analysed by pmoA-based terminal restriction fragment length polymorphism (T-RFLP), cloning, sequencing and quantitative polymerase chain reaction (qPCR), respectively. In addition, stable isotope probing of pmoA messenger RNA (mRNA) was performed to investigate the assimilation of 13C-acetate by the active methanotrophs in paddy soil.

Materials and Methods

Site Description

Soil was collected from a rice field at Sanjiang Experimental Station of Wetland Ecology, Chinese Academy of Sciences (47° 35′ N, 133° 31′ E), which is located in Sanjiang Plain in the eastern part of Heilongjiang province, China. The Sanjiang Plain, formerly the largest area of the freshwater wetlands in China, experienced incredible land-use changes during the past half century. Considerable areas of natural marsh (more than 3 million ha) have been reclaimed for agricultural purposes [23], and the area of rice fields increased especially since the 1990s and now accounts for one third of total cultivated area [24].

Soil was sampled after rice harvest in late autumn 2011. In total, six replicate cores (height, 20 cm; diameter, 5 cm) of the plow layer soil were collected, and the distance between two cores was about 10 m. The soils were mixed, air-dried, sieved (2-mm mesh size) and stored at room temperature. The soil was classified as Hydric Medihemists with the following basic characteristics: pH 5.8, organic matter of 4.0 %, total N of 1.0 g kg−1, total P of 0.54 g kg−1 and a texture of silty clay. The climatic conditions and agricultural practices, such as rice variety, water regime and fertilization, were described previously [25].

Experiment 1: Soil Incubation

Soil (10 g dry weight) was amended with 20 ml M1 medium in 50-ml serum bottles capped with butyl stoppers. The M1 medium was prepared according to published protocols [26]. The air headspace was supplemented with 10 % (about 100,000 ppm by volume) methane. All the bottles were incubated in the dark at 25 °C, with shaking at 120 rpm. After pre-incubating for 6 days, all the bottles were divided into three groups by supplying different substrates. The first group of bottles was continuously supplemented with 10 % methane until the end of incubation (referred as ME treatment). The second group was alternately supplemented with 10 mM acetate from days 6 to 12, days 16 to 21 and days 28 to 33, and 10 % methane in the remaining days (referred as AC treatment). The third group was the control relative to AC treatment by replacing acetate with the same volume of water (referred as CK treatment; Fig. 1). Each treatment was carried out in 12 replicate bottles. The original unincubated soil was sampled and referred to as ‘day 0’. On days 21 and 39, four bottles from each treatment were destructively sampled. Soil slurries were mixed thoroughly and then centrifuged at 5,000 rpm for 10 min. The precipitated soils were stored frozen at -20 °C until DNA extraction.

Fig. 1
figure 1

Methane oxidation rates of paddy soil under different incubation conditions. Bars represent standard errors (n = 4). ME continuously supplemented with methane, AC alternately supplemented with acetate and methane, CK alternately supplemented with water and methane

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During the incubation, the depletion of methane in the headspace was measured every 12 h by gas chromatography using a flame ionization detector (GC-FID) [27]. When the methane concentration dropped below 1 %, the bottle headspace was flushed with air and supplemented again to 10 % methane. Methane oxidation rates were calculated from the slopes of regression lines of methane depletion with time and expressed as micromoles CH4 h−1 per gram (dry weight) (gdw−1) of soil. The consumption of acetate in the slurry, with an average rate of 0.28 μmol h−1 gdw−1, was measured by high-performance liquid chromatography (HPLC) every 2 days. When acetate was consumed, the bottle was flushed with air and the new acetate was supplied to reach the concentration of 10 mM.

Experiment 2: Labelling Incubation

Incubation for stable isotope probing with acetate was performed by using the soil slurry from the above AC treatment on day 39. About 3 ml slurry was amended with a final concentration of 10 mM acetate (12C or 13C), dissolved with 1 ml M1 medium (4×) in 12-ml serum bottles. The fully labelled acetate (13CH3 13COOH, 99 % 13C) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Each labelling treatment was performed in triplicate. The bottles were incubated in the dark at 25 °C, shaking at 120 rpm. The consumed acetate was renewed to a final concentration of 10 mM every 2 days according to the results of a preliminary experiment, and the bottles were flushed with air at the same time. After labelling for 16 days, the soil slurry was sampled and frozen within 15 min using liquid nitrogen and stored at −80 °C until RNA extraction.

Nucleic Acid Extraction and SIP Fractionation

DNA was extracted by the Fast DNA Spin Kit for Soil (MP Biomedicals, Solon, USA) following the manufacturer’s instructions [27]. RNA was extracted using a bead-beating method following the detailed procedures described previously [28]. In brief, 0.5 g soil was extracted first with TPMS buffer once, and then twice with phenol-based lysis buffer. The pooled supernatants were further extracted with water-saturated phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol. The total nucleic acids were precipitated with cold ethanol, washed with cold 70 % ethanol and dissolved in TE buffer. The DNA in crude extracts was digested by RNase-free DNase (Takara, Dalian, China), and RNA was further purified by RNeasy® Mini Kit (Qiagen, Hilden, Germany). The quality and purity of DNA and RNA were checked by 1 % agarose gel electrophoresis and NanoDrop1000 spectrophotometry (NanoDrop Technologies, Wilmington, USA).

The purified RNA extracts from three replicates were pooled before gradient centrifugation. The gradient centrifugation of RNA was carried out in cesium trifluoroacetate (CsTFA) as described previously [29]. After centrifugation at 39,000 rpm for 65 h at 20 °C, the gradients were fractionated and the density of each fraction was determined by refractometry. RNA was precipitated with cold ethanol and 3 M sodium acetate (pH 5.2), washed with cold 70 % ethanol and finally resuspended in 30 μl of nuclease-free water. Then the PrimeScript™ 1st Strand complementary DNA (cDNA) Synthesis Kit (Takara) was used to synthesize the cDNA from RNA samples of each fraction by random priming according to the manufacturer’s instructions.

T-RFLP Analysis, Cloning, Sequencing and qPCR

DNA and cDNA samples were used for PCR amplification of fragments of pmoA using the primer set A189 and mb661. For the analyses of terminal restriction fragment length polymorphism (T-RFLP), the forward primer was labelled with 6-carboxyfluorescein (FAM). For cloning and sequencing, PCR amplification was performed using the same primers without a FAM label. T-RFLP, cloning and sequencing were performed according to previous protocols [11, 27]. Four clone libraries were constructed: three for pmoA genes (one soil on day 0 and two soils of AC and CK treatments on day 39, respectively) and one for pmoA transcripts (heavy fraction of labelled sample). In total, 177 randomly selected clones were sequenced. The operational taxonomic unit (OTU) cut-off value of 13 % based on nucleotide sequence identity was used to reflect methanotroph species [30]. One representative sequence from each OTU was chosen to construct the phylogenetic tree. The detailed procedures of phylogenetic analysis were described previously [27]. Sequences were deposited in the GenBank database under accession numbers KM924554 to KM924730.

The real-time (quantitative) PCR of pmoA fragments was performed in a 7500 real-time PCR system (Applied Biosystems) using the primer set A189 and mb661. Prior to qPCR, DNA preparations were diluted 10:1 to minimize possible inhibition. The DNA standard was prepared from purified plasmid DNA of a pmoA clone with a concentration ranging from 1.0 × 101 to 1.0 × 108 copies μl−1 [27]. The thermal cycles and fluorescence signal acquisition followed the protocol described previously [31]. Each measurement was performed in triplicate. For all assays, the amplification efficiency ranged between 89 and 94 % and r 2 values were 0.96 to 0.98.

Statistical Analysis

One-way ANOVA was performed to test for significant differences between treatments using SAS software (SAS Institute, Cary, USA). Other procedures related to data analyses have been described previously [11].

Results

Methane Oxidation Rate

In ME treatments, the methane oxidation rate increased to a peak on day 16, and then stabilized between 1.2 to 1.6 μmol h−1 gdw−1 until the end of the incubation (Fig. 1). In the CK treatment, the first and second stops of methane supply resulted in a significant decrease in methane oxidation rate, which recovered from day 35 to 37 to a level comparable to the ME treatment. The methane oxidation rate in the AC treatment was lower than in the ME and CK treatments. The rate decreased to almost zero on day 24. Surprisingly, the third addition of acetate did not cause a decrease. The rate maintained from 0.35 to 0.61 μmol h−1 gdw−1 after day 32, which was slightly higher than the rates from day 13 to 28. The oxygen concentration decreased rapidly after flushing with air, but was never lower than 54 μmol l−1, which is equivalent to 1 % (Fig. S1). Therefore, the incubation conditions remained oxic to prevent the possibility of anaerobic methane production by acetoclastic methanogenic archaea.

Quantification of pmoA Genes

The quantitative PCR of pmoA genes showed an obvious growth of methanotrophs during the incubation (Fig. 2). The highest growth was observed in the ME treatment, increasing from 6.8 × 108 copies gdw−1 on day 0 to 1.6 × 1011 copies gdw−1 on day 39. The copy numbers in AC and CK treatments also increased rapidly from days 0 to 21, but maintained stable from days 21 to 39. The differences among three treatments on day 21 were not significant (p > 0.05), although the copy number in the CK treatment was slightly lower. At the end of incubation (day 39), the copy numbers were significantly lower in AC and CK treatments than in the ME treatment (p < 0.01). In addition, the quantitative PCR of mcrA genes showed that the copy number decreased during the incubation, indicating that methanogens did not grow (Fig. S2).

Fig. 2
figure 2

Number of pmoA gene copies in paddy soil under different incubation conditions. Different letters on the same sampling day indicate a significant difference (p < 0.05)

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Methanotrophic Community Structure

The structure of methanotrophic community was investigated by a combination of T-RFLP analysis with cloning and sequencing of pmoA genes. In the pattern of original soil (day 0), the T-RF of 244 bp was found to be dominant with 80 % contribution, while other T-RFs of 79, 437 and 505 bp accounted for less than 20 % (Fig. 3). The 47 sequences in clone library for soil sample on day 0 were divided into six OTUs (Table 1; Table S1). Seventy-two per cent of sequences were related to the type II methanotroph Methylocystis (T-RF of 244 bp), and 28 % were related to type I methanotrophs. The type I groups consisted of Methylobacter (T-RF of 505 bp), Methylosarcina (T-RFs of 505 or 437 bp), Methylomonas (T-RFs of 505 or 437 bp) and Methylocaldum/Methylococcus (T-RF of 79 bp). Neither Methylocella- nor Methyloferula-related sequences were detected in a clone library of mmoX genes (Fig. S3), indicating that this paddy soil did not contain Methylocella or Methyloferula.

Fig. 3
figure 3

Structures of methanotrophic communities in paddy soil under different incubation conditions by T-RFLP-based analysis of pmoA genes

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Table 1 Analyses of four clone libraries of pmoA genes or transcripts
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The phylogenetic analysis of pmoA genes showed that sequences of OTU 1 were most closely related (99.7 % nucleotide sequence similarity, Fig. 4) to Methylocystis sp. LW2 which was isolated from the sediment of Lake Washington [32]. Among taxonomically described methanotrophs, Methylocystis echinoides IMET10491T displayed the highest identity with OTU 1 (96.3 % similarity of nucleotide sequence). M. echinoides IMET10491T, which was isolated from the sludge of sewage treatment plant [33], was able to slowly grow on acetate in the absence of methane [18]. An analysis by BLAST against pmoA sequences in the NCBI database (Table 2) indicated that OTU 1 were closely related (>99 % nucleotide sequence similarity) to numerous environmental sequences retrieved from paddy soils [37–40], rice roots [38, 41, 42] and other soils [43-45].

Fig. 4
figure 4

Phylogenetic tree of representative clone sequences of pmoA genes and transcripts retrieved from paddy soil. One representative sequence from each OTU was chosen to construct the phylogenetic tree

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Table 2 The environmental pmoA sequences highly identical to the sequences of OTU 1
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The methanotroph community compositions changed in the ME treatment after the incubation with methane (Fig. 3). The relative abundances of the 244 bp T-RF (Methylocystis) decreased whereas the T-RFs of 79 bp (Methylocaldum/Methylococcus) and 456 bp (Methylobacter) clearly increased. Similarly, the methane supplementation resulted in the decrease of the 244 bp T-RF (Methylocystis) and an increase of the 456 bp T-RF (Methylobacter) in the CK soil samples (Fig. 3, Table 1).

In contrast, the alternating supplementation with methane and acetate showed a different effect on methanotrophic compositions (Fig. 3). Compared with the T-RFLP pattern of the original soil, the relative abundance of T-RF of 244 bp in the AC treatment did not change much, but the T-RFs of 437 and 456 bp increased on days 21 and 39, respectively. The clone library for the soil sample from the AC treatment on day 39 showed that OTU 1 (Methylocystis, T-RF of 244 bp) became significantly dominant, accounting for 95 % of all sequences (Table 1).

Activity of Methanotrophs

Stable isotope probing of pmoA mRNA was performed for the 13C-acetate labelled soil and the unlabelled soil. The T-RFLP patterns among all fractions from the unlabelled soil sample were similar to each other (Fig. 5a). In contrast, the T-RFLP patterns from labelled soil differed significantly between heavy and light fractions. The T-RFs of 244 and 437 bp dominated in heavy and light fractions, respectively (Fig. 5b). In the clone library retrieved from the heavy fraction of labelled sample, the sequences belonging to OTU 1 (Methylocystis, T-RF of 244 bp) and OTU 4 (Methylomonas, T-RF of 505 bp) accounted for 72 and 28 %, respectively (Table 1).

Fig. 5
figure 5

T-RFLP fingerprints of pmoA transcripts retrieved from unlabelled (a) and labelled (b) samples in RNA-SIP

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Significant labelling of the bacterial 16S rRNA transcripts was observed, indicating the strong utilization of acetate directly or CO2 indirectly due to cross-feeding by soil bacteria (Fig. S4). Sequence analysis of 37 clones retrieved from the heavy fraction of the labelled sample showed that the active bacteria belonged to Pseudomonadales and Burkholderiales (Figs. S5 and S6). No sequences related to methanotrophs were observed, probably due to the insufficient numbers of clones or the low abundance of methanotrophs.

Discussion

To the best of our knowledge, this was the first study to investigate the activity and community structure of methanotrophs of Sanjiang Plain paddy soil. This paddy soil has two distinctive features, which are the highest latitude location and the lowest mean annual temperature among paddies around the world. Until now, most studies on paddy methanotroph diversity used the subtropical or tropical paddies as the model systems [43]. Therefore, the present study provides new information about the microbial methane oxidation of a paddy soil from a cold temperate zone. The methane oxidation rate of this paddy soil is relatively high and comparable to the rates of other subtropical paddy soils [6]. This indicated that the low mean annual temperature in Sanjiang Plain did not influence the potential activity of soil methanotrophs.

The analysis of community structure showed the presence of both type I and type II proteobacterial methanotrophs in Sanjiang Plain paddy soil, including members of the genera Methylobacter, Methylosarcina, Methylomonas, Methylocaldum/Methylococcus and Methylocystis. Such methanotrophic diversity has been detected in many other paddy soils [4, 27]. Furthermore, type II methanotrophs, especially the Methylocystis populations, were found to be more abundant than type I methanotrophs. Recently, a systematic study investigating methanotroph diversity in paddy soils from different temperate regions also revealed that Methylocystis are typically most abundant [43]. It seems that the predominance of type II methanotrophs is a common feature for paddy soils [9, 11], probably due to their special features [44]. For example, type II methanotrophs are able to form exospores or cysts during starvation and other forms of stress [45]. Therefore, they can survive adverse conditions and seem to be the major reservoir (or seed bank) of the methanotrophic community for the next season [46, 47]. This might also explain why type II methanotrophs are often detected in high abundance in the anoxic bulk soil [10].

Another reason for the predominance of type II methanotrophs is that they are able to use alternative carbon sources to support their growth when methane is limited [15]. The capacity to utilize multi-carbon compounds by facultative methanotrophs was first proven by the study of pure cultures. To date, there are four isolates of Methylocystis that are able to grow on acetate, namely Methylocystis bryophila H2s, Methylocystis heyeri H2T, M. echinoides IMET10491T [18, 48] and Methylocystis strain SB2 [19]. For example, Methylocystis strain H2s was shown to grow using acetate with a specific growth rate of 0.006 h−1 (equivalent to a doubling time of 115 h) [18]. Acetate utilization, as an important survival strategy for Methylocystis in the soils, has also been proven in environmental studies including peatlands [18] and fen soil [49]. The present study provides evidence of acetate utilization by Methylocystis in paddy soil. Firstly, we found that one genotype (OTU 1) closely related to M. echinoides IMET10491T was significantly enriched by acetate addition. In contrast, the type I methanotrophs Methylocaldum/Methylococcus and Methylobacter were enriched by the methane supplementation. Secondly, RNA-SIP of the pmoA gene transcripts revealed that OTU 1 was dominant in the heavy fraction of labelled samples, indicating that these Methylocystis-like methanotrophs actively assimilated the labelled acetate in paddy soil. Thirdly, the comparative analysis of environmental sequences from NCBI database showed that these uncultivated Methylocystis species (OTU 1-related) are widespread in paddy environments, including both bulk soil and rice root habitats.

Although the facultative methanotrophs were detected in paddy soil, the methanotrophic activity was significantly inhibited by acetate addition. Our results showed that the initial two additions of acetate did not influence the growth of methanotrophs, but significantly inhibited the methane oxidation activity. This is consistent with the findings from studies with pure culture [18], as well as with a forest soil [20] and fen soil [49]. This strongly suggests that methane is the preferred substrate for the facultative methanotrophs Methylocystis. Surprisingly, the paddy soil undergoing three periods of acetate addition still had methane oxidation activity, suggesting that the methanotrophs maintain the methane oxidation machinery by utilizing acetate in the absence of methane [18].

In summary, the present study showed that facultative methanotrophs belonging to the genus Methylocystis could use acetate as a secondary carbon source in paddy soil. This observation helps to explain the predominance of Methylocystis in paddy soils. Future efforts should aim to isolate these Methylocystis strains from paddy soil in order to expand our understanding of the diversity of facultative methanotrophs and their ecophysiological functions.