A 2-year field and incubation experiment was conducted to investigate δ13C during the processes of CH4 emission from the fields subjected to two water managements (flooding and drainage) in the winter fallow season, and further to estimate relative contribution of acetate to total methanogenesis (Fac) and fraction of CH4 oxidized (Fox) based on the isotopic data. Compared with flooding, drainage generally caused CH4, either anaerobically or aerobically produced, depleted in 13C. There was no obvious difference between the two in transport fractionation factor (εtransport) and δ13C-value of emitted CH4. CH4 emission was negatively related to its δ13C-value in seasonal variation (P<0.01). Acetate-dependent methanogenesis in soil was dominant (60–70%) in the late season, while drainage decreased Fac-value by 5–10%. On roots however, CH4 was mostly produced through H2/CO2 reduction (60–100%) over the season. CH4 oxidation mainly occurred in the first half of the season and roughly 10–90% of the CH4 was oxidized in the rhizosphere. Drainage increased Fox-value by 5–15%, which is possibly attributed to a significant decrease in production while no simultaneous decrease in oxidation. Around 30–70% of the CH4 was oxidized at the soil-water interface when CH4 in pore water was released into floodwater, although the amount of CH4 oxidized therein might be negligible relative to that in the rhizosphere. CH4 oxidation was also more important in the first half of the season in lab conditions and about 5–50% of the CH4 was oxidized in soil while almost 100% on roots. Drainage decreased Fox-value on roots by 15% as their CH4 oxidation potential was highly reduced. The findings suggest that water management in the winter fallow season substantially affects Fac in the soil and Fox in the rhizosphere and roots rather than Fac on roots and Fox at the soil-water interface.
Citation: Zhang G, Liu G, Zhang Y, Ma J, Xu H, Yagi K (2013) Methanogenic Pathway and Fraction of CH4 Oxidized in Paddy Fields: Seasonal Variation and Effect of Water Management in Winter Fallow Season. PLoS ONE 8(9): e73982. https://doi.org/10.1371/journal.pone.0073982
Editor: Wei-Chun Chin, University of California, Merced, United States of America
Received: March 15, 2013; Accepted: July 25, 2013; Published: September 12, 2013
Copyright: © 2013 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research work was financially supported by the “Strategic Priority Research Program–Climate Change: Carbon Budget and Relevant Issues” of the Chinese Academy of Sciences (XDA05020200), the National Natural Sciences Foundation of China (41071169), the Ministry of Science and Technology of China (S2012GR0080), and the Special Fund for Agro-scientific Research in the Public Interest (201103039). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Paddy fields are an important source of the greenhouse gas, methane (CH4), contributing to 5–19% of the total global CH4 emission . Proper water management is considered to be one of the most important options for regulating CH4 emission from paddy fields , . Generally, the fields are either intermittently irrigated or continuously flooded during the rice-growing season, and either drained without any irrigation except for rain water or kept flooded in the winter fallow season. Compared with continuous flooding, intermittent irrigation significantly decreases CH4 emission from rice fields during the rice-growing season by 40–70% –. Similarly, drainage, relative to flooding, in the winter fallow season not only prevents CH4 emission from the fields directly in the current season, but also sharply reduces CH4 emission indirectly during the following rice-growing season –. Although effects of water management in the winter fallow season on CH4 flux from the fields are considerably reported, its effect on the processes of CH4 emission, including CH4 production, oxidation and transportation, remains unclear. The stable carbon isotope technique, an important method for identifying processes of CH4 emission from rice fields, has been widely used through measuring carbon isotopic ratios –. In addition, it can be used to quantify contributions of various CH4 sources and provide information about carbon isotopes for global CH4 budget , . To our knowledge so far, very little study has been done on the measurement of stable carbon isotopes in the fields during the rice-growing season as affected by water management in the winter fallow season.
Methanogenesis is the precondition of CH4 emission from paddy fields and mainly occurs through two pathways. One is H2/CO2 reduction with the participation of specific hydrogenotrophic methanogens that use H2 or organic molecules as H donor (CO2+4H2 → CH4+2H2O). The other is acetate fermentation with the participation of acetotrophic methanogens (CH3COOH → CH4+ CO2). In general, the latter plays a more important role than the former in CH4 formation , . If δ13C-values of the CH4, CO2 and acetate involved in methanogenesis are measured, contributions of the two pathways can be estimated by using the stable carbon isotope technique , . Theoretically, acetate fermentation and H2/CO2 reduction accounts for 67% and 33%, respectively, of the total methanogenesis. Practically, relative contributions of the two pathways vary with rice cultivar, rice growth, water management, and environmental conditions, etc. , , , . During the rice-growing season, drainage can significantly enhance soil Eh, causing increase in oxidizing substances like Fe3+, sulphate and nitrate, and their inhibition of acetotrophic methanogens, thus reducing acetate-dependent methanogenesis , . In the winter fallow season, water management also significantly affects soil Eh, CH4 production and then CH4 emission from the fields during the following rice-growing season , but its impact on relative contributions of the two main pathways of methanogenesis remains poorly known.
CH4 oxidation, which occurs at the root–soil interface and soil–water interface, is very important to regulating paddy CH4 emission. By comparing CH4 emission from the field or CH4 production from aerobic incubation with methanogenesis in the strict anaerobic environment at the early stage, it was found that as much as 50–90% of the CH4 was oxidized before escaping into the atmosphere –. By using the stable carbon isotope method to quantify the fraction of CH4 oxidized in the paddy fields, recent studies in America and Italy indicated that it was less than 50% , , , . In China however, the fraction of CH4 that was oxidized in a paddy field under intermittent irrigation during the rice-growing season was measured by this means to be up to 80% . It was significantly higher than those in the fields under continuous flooding as above mentioned. Moreover, CH4 oxidation potential was relatively higher in intermittently irrigated paddy soil than in continuously flooded soil , which suggests that CH4 oxidation is highly impacted by water management during the rice-growing season. It is further indicated that oxidization of endogenous CH4 in the paddy fields seems to be more obvious in China, particularly in the fields that are intermittently irrigated during the rice-growing season. Although CH4 oxidation potential in paddy soil in a whole year has been reported , the percentage of CH4 oxidized in the field as affected by water management in the winter fallow season is still unknown.
Therefore, a 2-year field and incubation experiment was carried out in the paddy fields subjected to two types of water management (flooding and drainage) in the winter fallow season. Seasonal CH4 emission fluxes, CH4 in soil pore water and floodwater, CH4 in the aerenchyma of the plants, CH4 production and oxidation in fresh paddy soil and rice roots, and their respective δ13CH4-values during the rice-growing season were measured. The objectives of this study were: (1) to investigate impact of water management in the winter fallow season on CH4 production, oxidation and emission and their δ13CH4; and (2) further to evaluate its effect on pathways of CH4 production and fraction of CH4 oxidized in the fields by using the isotopic measurements.
Materials and Methods
Field Description and Experimental Design
With the authorization of the Institute of Agricultural Science, Zhenjiang City, the experiment was carried out at Baitu Town, Jurong City, Jiangsu Province, China (31°58′N, 119°18′E). Main features of the experiment field have already been described in detail before . The experiment was designed to have two treatments, three replicates each, i.e. winter fallow under continuous flooding (Flooding) and winter fallow without irrigation except for rain water (Drainage). Measurements of this study were performed during the 2008 and 2009 rice-growing seasons. Rice stubble and wild weeds were all removed from the experimental plots after rice harvest in the winter fallow season. For rice transplanting in the next rice-growing season, all the plots were ploughed the way the local farmers do. During the rice-growing season, they were continuously flooded and only drained for rice harvest. Rice seedlings (Cultivar “Oryza sativa L. Huajing 3”) were transplanted at their 3–4-leaf stage on June 22 and 26 in 2008 and 2009, respectively. Urea was applied at a rate of 300 kg N ha−1, of which 50% was done as basal fertilizer, 25% as tillering fertilizer, and 25% as panicle fertilizer. Both Calcium superphosphate and Potassium chloride were applied as basal fertilizer at a rate of 450 and 225 kg ha−1, respectively. For further details of the farming practices during the two years, please see Zhang et al. .
Field Sampling and Measuring
CH4 flux was observed using the static closed chamber method . To measure the flux, gas samples were collected at 4–7-day intervals using 18 mL vacuum vials. For determining isotopic signature (δ13C) of the emitted CH4, gas samples were taken at 10∼20–day intervals using 500 mL bags (Aluminium foil compound membrane, Delin gas packing Co., Ltd, Dalian, China) with a small battery-driven pump . The first sample was collected after the chamber was closed for 3–5 min, and the second at the end of the 2 h closure. Isotopic signature (S) of the emitted CH4 was calculated using the equation below:(1)where A and B stands for CH4 concentration (µl L−1) in the samples at the beginning and at the end, respectively, while a and b for the corresponding δ13CH4-values (‰) of the gas samples, separately. Simultaneously, soil redox potential (Eh) at the depth of 10 cm was measured, using Pt-tipped electrodes (Hirose Rika Co. Ltd., Japan) and an oxidation-reduction potential meter with a reference electrode (Toa PRN-41). Soil temperature at the depth of 10 cm was measured with a hand-carried digital thermometer (Yokogawa Meter and Instruments Corporation, Japan).
Soil pore water samples were collected using a Rhizon soil moisture sampler (10 RHIZON SMSMOM, Eijkelkamp Agrisearch Equipment, Giesbeek, Netherlands) . The samplers were installed (in triplicate) in the plots prior to rice transplanting and then left in the soil over the whole observational periods. Samples (∼5 mL) were firstly extracted using 18 mL vacuum vials to flush and purge the sampler before sampling. Then ∼10 mL of soil solution was drawn into another vial for further analysis. Simultaneously, 10 mL of floodwater was collected using a plastic syringe and then transferred in to an 18 mL vacuum vial. Finally, the pressure of all sampling vials was equilibrated by filling in pure N2 gas. After heavy shaking by hand, the airs in the headspace of the vials were directly analyzed for CH4 on the GC-FID, and their corresponding δ13CH4-values were determined using the isotope ratio mass spectrometer. CH4 concentrations (CCH4) in pore water and floodwater were calculated using the following equation:(2)where m stands for mixing ratio of CH4 in the headspace of a vial (µL L−1), MV for volume of an ideal gas (24.78 L mol−1 at 25°C), GV for volume of the gas headspace of the vial (L), and GL for volume of the liquid in the vial (L).
Samples of the gas in the aerenchyma of and emitted from the plants were taken using specially designed PVC bottomless pots . The pot, 30 cm in height and 17 cm in diameter, was designed to have a water-filled trough around its top, avoiding any possible gas exchange during the sampling times. A PVC plate (18 cm in diameter) with a hole (the diameter could be adjusted to the growing plant) in the center was placed on top of each pot, allowing the plant to grow through the hole and keep divided into two parts. Then, one plant inside the pot was cut right above the plate while the other remained intact as the control. Finally, chambers (30×30×100 cm) were laid on the pots, and gas samples in the headspace of the chambers were collected simultaneously with a small battery-driven pump.
Triplicate soil cores were collected from each experimental plot using a stainless steel corer (7 cm diameter×25 cm length) and then prepared into mixture . Samples (in triplicate) from the mixture, about 50 g (dry weight) each, were taken and promptly transferred into 250-mL Erlenmeyer flasks separately, and turned into slurries with N2-flushed de-ionized sterile water at the ratio of 1∶1 (soil/water). During the whole process, the samples were constantly flushed with N2 to remove O2 and CH4, and the flasks containing these samples were sealed for anaerobic incubation. Some other flasks with air headspace were sealed directly for aerobic incubation. They were all stored in N2 at 4°C for further analysis within 8 h. A small portion of the soil sample was dried for 72 h at 60°C for determination of isotopic composition of the organic carbon.
Complete rice plants together with roots were carefully collected from the experimental plots at each main rice growth stage, i.e. tillering (TS), booting (BS), grain-filling (FS) and ripening (RS) stages, in 2009 . The roots were washed clean with N2-flushed demineralized water and cut off from the green shoots at 1–2 cm from the root with a razor blade. The fresh roots, 20 g each portion, were then put into flasks, separately, for further preparation and processing in the same way as for the soil. The shoots were dried at 60°C for 72 h for dry weight measurement and then stored at room temperature for determination of isotopic composition of the organic carbon.
Fresh Soil and Roots Incubations
CH4 production potentials were measured for the paddy soil and rice roots under anaerobic incubation. The flasks were flushed with N2 consecutively for six times through double-ended needles connecting a vacuum pump to purge the air in the flasks of residual CH4 and O2. Simultaneously, methanogenesis was determined aerobically using flasks with air headspace directly. They were subsequently incubated at a temperature the same as measured in the field for 50 h in darkness. Gas samples were collected twice with a pressure lock syringe, and analyzed 1 h and 50 h later after the flasks were heavily shaken by hand, for CH4 on the GC-FID. CH4 production was calculated using the linear regression of CH4 increasing with the incubation time.
CH4 oxidation potentials were determined for the paddy soil and rice roots under aerobic incubation with high CH4 concentration supplemented, using equipment the same as described above. Firstly, pure CH4 was injected into each flask to make a high concentration inside (∼10000 µL L−1). Then, the flasks were incubated in dark under the same temperature as measured in the field and shaken at 120 r.p.m. CH4 depletion was measured by sampling the headspace gas in the flask after vigorous shaking for subsequent GC-FID analysis. The first sample was collected generally 30 min after pure CH4 was injected. Samples were then taken in 2–3 h intervals during the first 8 h of the experiment. They were left in the flasks over night and measured the next day in 2 h intervals again. CH4 oxidation was calculated by linear regression of CH4 depletion with incubation time.
CH4 was quantified using the gas chromatograph (GC) equipped with a flame ionization detector (FID) . The isotopic composition (δ13C) of CH4 and CO2 was determined with a Finnigan MAT-253 Isotope Ratio Mass Spectrometer (IRMS, Thermo-Finnigan, Bremen, Germany) using the continuous flow technique , . The IRMS had a fully automatic interface for pre-GC concentration (Pre-Con) of trace gases, and the precision of repeated analyses was ±0.196‰ (n = 9) with 2.02 µL L−1 CH4 injected. Gas samples were first blown into the chemical trap with Mg(ClO4)2 and ascarite by He flow (20 mL min−1). Over 99.99% of the CO2 and H2O in the samples was absorbed and removed. CH4 in the samples was then converted into CO2 in a combustion reactor at about 1000°C. Subsequently, it was flowed into the freezing traps with liquid nitrogen (–196°C) and the GC for further separation. The separated gases were finally transferred into the mass spectrometer for δ13C determination. The dried soil and plant samples were analyzed for carbon isotope composition with a Finnigan MAT-251 Isotope Ratio Mass Spectrometer (Thermo Finnigan, Bremen, Germany). Soil organic carbon contents were measured by wet oxidation using dichromate in acid medium followed by the FeSO4 titration method.
Isotope ratios are expressed in the standard delta notation:(3)where R stands for 13C/12C of the sample and the standard, respectively, using PDB carbonate for the standard. Carbon isotope fractionation factor during acetate fermentation (εacetate/CH4) or H2/CO2 reduction (αCO2/CH4) for methanogenesis was defined by Hayes :
Relative contribution of acetate to total CH4 (Fac) was calculated using the following mass balance, assuming that acetate fermentation and H2/CO2 reduction were the only sources of methanogenesis in the rice fields -:(6)(7)where δ13CH4 stands for δ13C value of total CH4. In addition, the fraction of CH4 that was oxidized (Fox) in the fields was estimated using the equation given by Stevens and Engelkemeir  and Tyler et al. :(8)where δ13CH4(original) stands for carbon isotopic signature of the primarily produced CH4, δ13CH4(oxidized) for carbon isotopic signature of the residual CH4 after oxidization, of which the calculation was done using a semi-empirical equation :(9)and αox stands for isotope fractionation factor due to CH4 oxidation by the methanotrophs, and εtransport for isotope fractionation factor due to CH4 transport by the plants.
Statistical analysis was done using the SPSS 18.0 software for Windows (SPSS Inc., Chicago). Differences between the two treatments in mean (n = 3) CH4 concentration, mean CH4 production and oxidation potentials, and mean soil Eh were determined through one-way analysis of variance (ANOVA) and least significant difference (LSD) test. Relationships between CH4 fluxes and emitted δ13CH4 (n = 18), between mean CH4 production potential and soil Eh (n = 11), and between CH4 oxidation potential and soil temperature (n = 11) were assessed using correlation analysis. Statistical significant differences and correlations were set at P<0.05.
CH4 Emission and δ13C
CH4 emissions (Fig. 1a, e) were significantly higher from flooded fields than from drained fields as had been reported before . Different variation patterns were observed in the δ13C of the emitted CH4 in 2008 and 2009 seasons (Fig. 1b, f). Generally, the emitted CH4 tended to be 13C-enriched in 2008 with its δ13C-value increased from –69 to –51‰ in Treatment Flooding, and from –65 to –47‰ in Treatment Drainage (Fig. 1b). However, more complicated changes were observed in 2009, showing that the emitted CH4 was relatively enriched in 13C at the beginning and at the end of the season, and relatively 13C-depleted in the middle of the season (Fig. 1f). However, little difference was found between Treatments Flooding and Drainage, with δ13C-values being in the range of –68 ∼ –48‰ and –71 ∼ –53‰, respectively (Fig. 1b, f, P>0.05). Although more measurements were performed in 2009 than in 2008, the mean δ13C-value seemed to be more positive in 2008 (–58 ∼ –55‰) than in 2009 (–62 ∼ –61‰). Notably, negative relationship was observed between CH4 flux and δ13C in the seasonal variation in 2008 (r = –0.695, P<0.01) and 2009 (r = –0.546, P<0.01).
(a, b, c and d) 2008, (e, f, g and h) 2009. TS, BS, FS and RS represent tillering, booting, grain-filling and ripening stages, respectively. Bars represent standard errors (n = 3).
Significant difference in soil Eh was observed between the two treatments (Fig. 1c, g, Table 1). Soil Eh was very close to 0 mV at the beginning of the season and remained much lower in Treatment Flooding than in Treatment Drainage throughout the two seasons, with the averaged value of –165 and –88 mV in 2008, and –153 and –26 mV in 2009, respectively. Soil temperature at the depth of 10 cm generally peaked around D25 (25 days after rice transplanting) and then gradually declined till the end of the season (Fig. 1d, h), fluctuating within the range from 16 to 30.1°C in 2008 and from 17.2 to 29.7°C in 2009, and being averaged 24.3 and 24.4°C, respectively.
CH4 Concentration and δ13C
CH4 concentration in pore water for Treatment Flooding was relatively high (200–400 µmol L−1) at the beginning of the season, dropped subsequently to 20–200 µmol L−1 and then turned upwards again to 350–450 µmol L−1 at the end of the season in both 2008 and 2009 (Fig. 2a, c). For Treatment Drainage however, CH4 concentration decreased gradually from 300 to 200 µmol L−1 during the 2008 season (Fig. 2a), whereas it was the highest (∼200 µmol L−1) in the middle and the lowest (<50 µmol L−1) at the beginning and the end of the 2009 season (Fig. 2c). The averaged CH4 concentration during the two seasons was generally higher in Treatment Flooding than in Treatment Drainage (Table 1). δ13C-value of the CH4 was relatively stable during the 2008 season though it increased and then slightly decreased (Fig. 2b). As a whole, CH4 was much more 13C-enriched in Treatment Flooding (–65‰) than in Treatment Drainage (–67‰) over the 2008 season (Fig. 2b, P<0.05). In the 2009 season however, δ13C-value fluctuated sharply within the range from –65 to –55‰ to –70‰ or so (Fig. 2d). No obvious difference in mean δ13C-value (∼ –60‰) was observed between the two treatments in 2009 (Fig. 2d, P>0.05).
(a, b) 2008, (c, d) 2009. TS, BS, FS and RS represent tillering, booting, grain-filling and ripening stages, respectively. Bars represent standard errors (n = 3).
CH4 concentration in floodwater of the field in 2009 was measured simultaneously. No more than 7 µmol L−1 of CH4 was detected though little data were obtained (Fig. 2c). On the other hand, CH4 in floodwater became more and more 13C-enriched towards the end of the season, with the δ13C-value increased from –50 to –40‰ (Fig. 2d). Little difference in δ13C-value was observed as well between Treatments Flooding and Drainage (Fig. 2d, P>0.05). Compared with porewater CH4, floodwater CH4 was much more enriched in 13C (Fig. 2d, P<0.05).
Plants Emitted and Aerenchymatic CH4 and δ13C
To quantify stable carbon isotope fractionation during the CH4 emitted through the aerenchyma of the plants, δ13C-values of the emitted CH4 and aerenchymatic CH4 were measured simultaneously. On the three sampling days during the 2009 season, the emitted CH4 was relatively stable with its δ13C-value stable around –60‰ (Table 2). The aerenchymatic CH4 as expected, was significantly 13C-enriched compared to the emitted CH4, with the δ13C-values varying in the range of –51 ∼ –42‰, and being about –47‰ on average for the two treatments (Table 2). As a result, the isotope fractionation factor due to CH4 transport (εtransport) was determined to be in the range from –16 to –11‰ in Treatment Flooding and from –14 to –12‰ in Treatment Drainage. As a whole, no obvious difference in mean value of εtransport (∼ –13‰) was observed between the two treatments (Table 2, P>0.05).
CH4 Production Under Anaerobic Incubation and δ13C
CH4 production potentials of the slurries of paddy soil were measured during the 2008 and 2009 rice seasons (Fig. 3a, d). Methanogenesis started more quickly and became more intense in Treatment Flooding than in Treatment Drainage over the two seasons (Fig. 3a, d), peaked around D40–60 for the former and around D80 for the latter. Mean production potential was significantly higher in Treatment Flooding than in Treatment Drainage during the two seasons (Table 1, P<0.05). The produced CH4 was relatively stable in δ13C (∼ –60‰) in Treatment Flooding while fluctuated sharply (from –72 to –55‰) in Treatment Drainage in 2008 (Fig. 3b). In 2009 however, it was gradually enriched in 13C for the two treatments, with δ13C-value ranging from –70 to –60‰ (Fig. 3e). In addition, the mean δ13CH4-value in Treatment Flooding appeared to be slightly more positive than that in Treatment Drainage over the two seasons, varying in the range of –63 ∼ –58‰ and –66 ∼ –63‰, respectively. The produced CO2 became isotopically heavier step by step, causing δ13C-value to decrease from –20 ∼ –15‰ at the beginning of the season to around –10‰ at the end of the season, and it was relatively more 13C-enriched in Treatment Flooding than in Treatment Drainage over the two seasons (Fig. 3c, f, P>0.05).
(a, b and c) 2008, (d, e and f) 2009. TS, BS, FS and RS represent tillering, booting, grain-filling and ripening stages, respectively. Bars represent standard errors (n = 3).
Abundant methanogenesis was measured on the fresh rice roots under anaerobic incubation in 2009 (Fig. 4a). The production of CH4 increased sharply and peaked around D60, just like the soil (Fig. 3d). Then it decreased gradually till the end of the season. As a whole, it was significantly higher in Treatment Flooding than in Treatment Drainage (Fig. 4a, Table 1, P<0.05). Similar to CH4 produced in the soil, CH4 produced on the roots was depleted in 13C at the beginning of the season (Fig. 4b). Subsequently, it became more 13C-enriched, with its δ13C-values ranging from –90 to –75‰. No significant difference was observed in mean δ13C-value between Treatment Flooding (–83‰) and Treatment Drainage (–81‰). However, it was much more negative compared to the CH4 produced in the soil in δ13C-value (Figs. 3e and 4b, P<0.01). The δ13C-value of produced CO2 ranged from –22 to –17‰ over the two seasons and no obvious difference was observed between the two treatments (Fig. 4c, P>0.05).
CH4 Production Under Aerobic Incubation and δ13C
Less than 0.3 µgCH4 gsoil−1 d−1 was produced in the soil under aerobic condition over the 2009 season (Fig. 5a), and 1.0–1.5 µgCH4 groots−1 d−1 was on the roots at the beginning of the season and below 0 µgCH4 groots−1 d−1 at the end of the season (Fig. 5c). The produced CH4 was very stable over the season both in the soil and on the roots, with δ13C-values around –58 ∼ –55‰ and –44 ∼ –41‰, respectively. Opposite to the CH4 produced under anaerobic condition, it was significantly more enriched in 13C on the roots than in the soil (Fig. 5b, d, P<0.01). Generally, the δ13C-value was more positive in Treatment Flooding than in Treatment Drainage (Fig. 5b, d). In addition, the CH4 produced under aerobic condition was significantly 13C-enriched relative to that produced under anaerobic condition, especially those from the roots (Figs. 3e, 4b and 5b, d, P<0.01).
CH4 Oxidation Under Aerobic Incubation Amended with High CH4 Concentration
Similar variation patterns of the CH4 oxidation potentials in the paddy soil during the 2008 and 2009 seasons were observed, showing a peak at the beginning of the season and a steep slope till the end of the season (Fig. 6a, b). Although the potential was relatively lower in Treatment Flooding than in Treatment Drainage in the middle of the season but slightly higher both at the beginning and at the end of the season (Fig. 6a, b), no significant difference was observed between the two treatments in mean of the potential (Table 1, P>0.05). Notably, a significant positive relationship was observed between CH4 oxidation potential and soil temperature in temporal variation (r = 0.703–0.859, P<0.05). Intensive oxidation signal on the fresh roots was observed, which was also the strongest (400–600 µgCH4 groots−1 d−1) at the beginning of the season and declined to the lowest (150–400 µgCH4 groots−1 d−1) at the end of the season (Fig. 6c). Throughout the 2009 season, CH4 oxidation potential on the roots was significantly higher in Treatment Flooding than in Treatment Drainage (Fig. 6c, P<0.05).
Organic Carbon in Soil and Plant Samples
During the 2008 season, the content of organic carbon in the soil was 1.02±0.08% in Treatment Flooding and 1.11±0.05% in Treatment Drainage, and it seemed to increase during the 2009 season, reaching 1.65±0.01% and 1.83±0.10%, respectively. Soil organic carbon in Treatment Drainage was very stable in δ13C (–27.9‰) during the two rice seasons, whereas it was slightly 13C-enriched in Treatment Flooding, with δ13C-value increasing from –28.1‰ in 2008 to –27.0‰ in 2009. Organic carbon in the plant samples showed little change throughout the 2009 season, with δ13C-value of –28.9‰, –29.2‰ and –28.7‰ on D27, D66 and D108, respectively, although it was slightly lighter in contrast to the organic carbon in the soil.
Effects on Stable Carbon Isotopes
The processes of CH4 emission involved in CH4 production, oxidation and transportation in the fields were well identified by measuring stable carbon isotopes (Fig. 7). In paddy fields, besides the applied organic fertilizers, plant photosynthesis and degradation of soil organic carbon are the two most important substrate sources for methanogenesis . As substrates for CH4 production, δ13C-value of organic carbon in the plant samples (–29‰) seemed to be slightly negative than that in the soil samples (–27‰) (Fig. 7). Previous observations also showed that organic carbon was slightly lighter in the plant than in the soil , , . Intensive carbon isotope fractionation generally happens when methanogenic precursors form CH4. Around 10–20‰ occurs during CH4 production through acetate fermentation while 50–70‰ during CH4 production through H2/CO2 reduction , . As a consequence, CH4 from the former (–60 ∼ –50‰) is usually more positive than that from the latter (as negative as –110‰) . The CH4 produced in the soil was more 13C-enriched in Treatment Flooding than in Treatment Drainage (Fig. 3b, e) and also more (–65‰) than that on the roots (–80‰) in both treatments (Fig. 7). This shows that flooding, compared with drainage in the winter fallow season, increased the relative contribution of acetate to CH4 production and that aceticlastic methanogenesis in the soil was more important than that on the roots (Fig. 8). Early anaerobic measurements indicated that CH4 from the roots was more depleted in 13C than that from the soil , .
Stable carbon isotopes in the soil and plant organic carbon and CH4 isotopic compositions in the processes of CH4 emission from the paddy fields and in the lab conditions. The δ13CH4-value of each component was given in the range of isotopic variation during the 2009 rice season.
(a) 2008, (b and c) 2009. Fac was calculated with Eq. (7) using 1.079 for αCO2/CH4 and –43‰ for δ13CH4 (acetate). TS, BS, FS and RS represent tillering, booting, grain-filling and ripening stages, respectively. Bars represent standard errors (n = 3).
In rice-based ecosystems, the produced CH4, except for the portions oxidized and emitted into the atmosphere, is temporarily retained in the soil as entrapped CH4 and dissolved CH4 in soil pore water . As mainly in the form of bubbles, CH4 in soil pore water probably remains unoxidized and is usually considered to be the original CH4 produced in the field in many reports , , . However, Krüger et al.  found that CH4 in soil pore water poorly represented the produced CH4. In the present study, it might be partially oxidized as well in the rhizosphere during the periods of D30–60 in the 2009 season (Fig. 6d), though its mean δ13C-value lingered around –60‰ and close to that of the CH4 produced in anaerobic soil over the season (Fig. 7). When porewater CH4 is released through the soil-water interface in paddy fields, it will be considerably oxidized, leaving the remainder temporarily in the floodwater. Since 12CH4 is consumed faster than 13CH4 by soil microbes, the residual CH4 is then 13C-enriched . As a consequence, floodwater CH4 (–45‰) was more 13C-enriched than porewater CH4 (–60‰). The observation of δ13C-value of the CH4 produced in aerobic soil being more positive than that in anaerobic soil (Fig. 7) further demonstrates that CH4 oxidation is intensive at the soil-water interface. In addition, rice roots can excrete O2 thus forming an important CH4-oxidizing zone in the rhizosphere. What is more, fresh rice roots per se have a strong CH4 oxidation capacity , , . CH4 aerobically produced on the roots appeared to be more enriched in 13C (–45‰) than that in the soil (–55‰). It suggests that rice roots in the rhizosphere may be more important than the soil per se as driving force for CH4 oxidation, thus causing more 12CH4 consumed and leaving more 13CH4 remained (Fig. 7).
Aerenchymatic CH4 (∼ –47‰) was similar to oxidized CH4 in δ13C-value (Fig. 7), which demonstrates that it has been strongly oxidized in the rhizosphere. In Italian paddy fields, Krüger et al.  also found that it stayed around –50‰ throughout the rice-growing season. After being emitted through transportation of the plants, aerenchymatic CH4 was much heavier than emitted CH4 (Fig. 7), due to the fact that 12CH4 was transported from the plants at a faster rate than 13CH4 . By subtracting δ13C-value of aerenchymatic CH4 from δ13C-value of emitted CH4 the transport fractionation by the plants is quantified –. In theory, the transport fractionation is relatively small due to small CH4 transport capacity of the plants at the beginning of the season. It gets strengthened together with the growth of the plants during the middle of the season but weakens again till the end of the season because of aging of the roots and plants. Consequently, value of fractionation (εtransport) was the lowest during the middle of the season, and relatively high at the beginning and the end of the season because it was shown as negative (Table 2). Moreover, it was averaged around –13‰, suggesting that water management in the winter fallow season has little effect on CH4 transport fractionation during the following rice-growing season. Similar εtransport was also observed in other field experiments –.
The δ13C-value of emitted CH4 fluctuated largely during the 2008 and 2009 rice seasons (Fig. 1b, f), and they were negatively related to CH4 emission in seasonal variation (r = –0.695 ∼ –0.546, P<0.01). An analogous relationship between them was also observed in other experiments , , which was considered to be the combined effect of CH4 production, oxidation and transport in the fields , , . Although water management in the winter fallow season played a key role in CH4 emission from the rice fields (Fig. 1a, e), it had little impact on δ13C-value of emitted CH4 (Fig. 1b, f). For the two seasons, mean value was ∼ –60‰, being in the range of the measurements in previous report . Compared with flooding, drainage had CH4 relatively more depleted in 13C (Fig. 3b, e), but the CH4 would become enriched in 13C again after it was oxidized because Fox-value in the latter was 5–15% higher (for detailed description, please see Section Effects on CH4 oxidation below). In addition, there was no obvious difference in εtransport between the two treatments (Table 2). Therefore, the 13C-depleted CH4 in Treatment Drainage was supposed to be offset by the higher fraction of CH4 oxidation, thus making the δ13C-value of emitted CH4 from the two treatments similar.
Effects on CH4 Production
Previous studies demonstrated that water management in the winter fallow season significantly affected CH4 production during the following rice-growing season , . In the present study, it showed an important effect on CH4 production of the fields by significantly affecting soil Eh. Methanogens are a kind of extreme anaerobic bacteria, which produce CH4 under strict reductive conditions. Compared to the fields flooded in the winter fallow season, the fields drained were probably much lower in population and activity of methanogens – and it generally took a longer time for methanogens to revive during the following rice-growing season . Therefore, drainage delayed and decreased CH4 production in soil by significantly increasing soil Eh (Figs. 1c, g and 3a, d, Table 1). On roots as well, the lower the soil Eh, the higher the CH4 production (Table 1) because roots get to age and decay faster if they are constantly under a stronger reductive condition , . A significant negative correlation between mean CH4 production and soil Eh (Table 1, r = –0.805, P<0.01 for soil and r = –0.994, P<0.01 for roots, respectively) better demonstrated that soil Eh significantly affected by water management in the winter fallow season was a key factor that influenced CH4 production. CH4 concentration in soil pore water being generally lower in Treatment Drainage than in Treatment Flooding (Table 1) further showed that drainage decreased CH4 production in the fields.
In paddy fields, CH4 mainly comes from acetotrophic and hydrogenotrophic methanogenesis. Methanol-dependent methanogenesis may possibly be another contributor to the total CH4 production, though, insignificant . Relative contribution of acetotrophic methanogenesis (Fac) to the total CH4 production can be calculated by following Eq. (7) if a fractionation factor of αCO2/CH4 = 1.079 is used for CO2-dependent methanogenesis and δ13CH4 (acetate) = –43‰ is for acetate-dependent methanogenesis based on the following reports , . In an Italian paddy soil, Fey et al.  found that αCO2/CH4, decreasing with increasing temperature, was 1.083 at 10°C, 1.079 at 25°C, and 1.073 at 50°C, which was in good agreement with the relationships in marine sediment  and methanogenic cultures . Therefore, αCO2/CH4 = 1.079 was applied because the temperature during the two seasons varied in the range of 20–30°C with an average of 24°C. On the other hand, Fey et al.  demonstrated that δ13CH4 (acetate) increased with increasing temperature, e.g., from –50 ∼ –46‰ at 10°C to –45 ∼ –36‰ at 25°C, and to –43 ∼ –31‰ at 37°C. Moreover, the δ13CH4 (acetate)-values of –43 ∼ –36‰ have even been applied considerably to experiments in the fields during the rice-growing season –, . Due to lack of measurements, a constant value of –43‰ was hence used in the present study for better comparison with these reports. What is more important, it was more reasonable and suitable because δ13C-value of the soil organic carbon–substrate for methanogenesis, in this study was similar to those observed before , , . Although they might be different in microbe habitats and varied with temperature and rice growth , , , the same values of αCO2/CH4 and δ13CH4 (acetate) above mentioned have also been used , , .
The findings show that variation of Fac-value in paddy soil during the 2008 rice season was similar to that during the 2009 rice season in pattern. That is, acetate-dependent methanogenesis dominated in the late season, while it was not so much important in the mid season, with Fac-value being over 60–70% and less than 40%, respectively (Fig. 8a, b). In Italian paddy fields, measurements also show that it was dominant at the end of the season . Water management in the winter fallow season had an important impact on methanogenic pathways of paddy soil during the following rice-growing season. Generally, CH4 from acetate cleavage dominated in Treatment Flooding during the two rice seasons, having a mean Fac-value of 53–65%, which was 5–10% higher than in Treatment Drainage (Fig. 8a, b). Drainage increased production of oxidants, such as Fe3+ or sulphate, along with the increase in soil Eh , . As a result, the growth and activity of methanogens was probably out-competed by iron- or sulphate-reducing bacteria , . More importantly, acetotrophic methanogens seemed to be inhibited to a larger extent than hydrogenotrophic methanogens , . This suggests that soil Eh is an important indicator of pathways of methanogenesis in paddy fields–the higher the soil Eh, the more inhibited the acetotrophic methanogens, and the less the contribution of acetate to the total methanogenesis. In the present study therefore, drainage in the winter fallow season significantly increased soil Eh (Fig. 1c, g) and obviously decreased methanogenesis in paddy soil compared to flooding (Fig. 3a, d), and hence the contribution of acetate-dependent methanogenesis, probably ascribed to the fact that acetate-utilizing methanogens was more inhibited by any increase in soil Eh (Fig. 1c, g) . Intermittent irrigation during the rice-growing season significantly reduced the contribution of acetate to CH4 production, of which the finding could further verify this point of view .
The relative contribution of acetate-dependent methanogenesis on rice roots was similar to that in paddy soil in 2009, which was the lowest (almost 0%) in the mid season but rose up to the highest (∼40%) at the end of the season (Fig. 8c). In total, Fac-value was 1–32% in Treatment Flooding and 0–38% in Treatment Drainage, being much lower than that in the soil (Fig. 8). It indicates that methanogenesis on fresh rice roots is mostly from H2/CO2, and it is little affected by water management in the winter fallow season. Previous reports also show that Fac-value of rice roots was less than 40% in most of the season . In an incubation experiment with rice roots D75–80 old, Conrad et al.  found that CH4 mainly came from H2/CO2-dependent methanogenesis as well throughout the entire observation, with an average Fac-value of 47%. Compared with that of soil, the relative contribution of acetate to the total methanogenesis on the roots was lower by approximately 30% (Fig. 8), which is likely attributed to the difference in population of their dominant methanogens –. More exact measurements using stable isotope probing techniques have further demonstrated that CH4 production on roots depends mainly on H2/CO2 reduction triggered by RC-I methanogens (Rice Cluster I Archaea) , . On the other hand, organic carbon slightly lighter in plant samples than in soil samples might be a possible reason for Fac-value being much lower in paddy soil than on rice roots.
Effects on CH4 Oxidation
CH4 oxidation in soil seemed to be highly influenced by soil temperature rather than water management in the winter fallow season. Firstly, no significant difference was observed between flooding and drainage in mean oxidation potential during the 2008 and 2009 seasons (Table 1). Secondly, it varied with soil temperature (Figs. 1d, h and 6a, b), and a positive relationship was observed over the two seasons (r = 0.703–0.859, P<0.05), which is in good agreement with the previous report . An appropriate soil temperature favors growth of methane-oxidizing bacteria, thus enhancing their capacity of CH4 oxidation . The higher the soil temperature within the range of 12.5–34.8°C, the higher the CH4 oxidation rate , which is consistent with our observations.
Considerable measurements on fresh roots have shown that the roots per se have a high CH4 oxidation capacity , , . In the present study, CH4 oxidation on the roots was the strongest at the beginning of the season and weakened later on (Fig. 6c), being in agreement with the previous reports , . Drainage compared to flooding in the winter fallow season significantly decreased CH4 oxidation potential on the roots (Fig. 6c), probably attributed to the effect of flooding highly increasing CH4 production (Fig. 4a). Higher concentration of CH4 stimulated growth and activity of the methanotrophs on the surface of the roots, thus raising their CH4 oxidation capacity .
The fraction of CH4 oxidation (Fox) can be quantified by measuring δ13C-value of CH4 from various compartments of the paddy fields with a special model in case some parameters (αox and εtransport) are already available –. The potential shift in the carbon isotopes during the CH4 oxidation (fractionation factor αox = 1.025–1.038) was firstly determined in methanotrophs enriched cultures  and then considerably in landfill cover soils at a temperature of about 25°C –. Interestingly, the value of 1.025–1.038 has been widely applied to field conditions –, ,  though the knowledge of αox in paddy soil is still incomplete. Very recently, we have found αox = 1.025–1.033 at 28.3°C in a Chinese paddy soil . Consequently, the value of 1.038 was used in the present study due to the similar temperature during the seasons, and more reasonable results would be obtained (Fig. 9). On the other hand, the transport fractionation factor εtransport was equivalent to the difference in 13C between emitted and aerenchymatic CH4 (Table 2), ranging from –16 to –11‰ in the 2009 season. In 2008 however, no corresponding measurements were performed. Nevertheless, the averaged value of –13‰ in 2009 was applied to the 2008 field data (Fig. 9), because it was also very close to previous observations –. Since fractionation factors (αox and εtransport) are influenced by temperature, microbes, soil property, and rice growth , , more attention thereby need further be paid to getting reliable and exact values of CH4 oxidation in paddy fields.
Fox in (a) and (b) was calculated with Eq. (8) using 1.038 for αox, δ13C-values of CH4 anaerobically produced in soil (Fig. 3b, e) for δ13CH4 (initial), and δ13C-values of emitted CH4 (Fig. 1b) minus –13.0‰ for both treatments in 2008 but (Fig. 1f) minus –13.3‰ for flooding and –12.8‰ for drainage in 2009 for δ13CH4 (final). Fox in (c) and (d) was calculated in 2009 with Eq. (8) using 1.038 for αox, δ13C-values of CH4 anaerobically produced in soil (Fig. 3e) and on roots (Fig. 4b) for δ13CH4 (initial), and δ13C-values of CH4 aerobically produced in soil (Fig. 5b) and on roots (Fig. 5d) for δ13CH4 (final). TS, BS, FS and RS represent tillering, booting, grain-filling and ripening stages, respectively. Bars represent standard errors (n = 3).
Similar to the potentials of CH4 oxidation, the fraction of CH4 oxidized in the rhizosphere was relatively high (as high as 60–90%) in the first half of the rice growth period during the 2008 and 2009 seasons and relatively low (∼10–30%) in the remainder periods (Fig. 9a, b). In Italian paddy fields, measurements also show that CH4 oxidation was very important at the beginning of the season but became slight later, with Fox-value decreasing rapidly from approximately 40 to 0% , , . Under unfertilized microcosms, Conrad and Klose  obtained that Fox-value decreased from about 15% in the beginning to about 5% at the end, which was probably attributed to nitrogen-limitation of the methanotrophs , , . On the whole, mean value of Fox was 35–55% in Treatment Drainage, being 5–15% higher than that in Treatment Flooding during the 2008 and 2009 seasons (Fig. 9a, b). It suggests that compared to flooding in the winter fallow season drainage can increase the proportion of CH4 oxidized during the following rice-growing season. Probable reason was that drainage significantly decreased CH4 production (Fig. 3a, d) while it did not simultaneously affect CH4 oxidation in the field (Fig. 6a, b).
When CH4 in soil pore water passed through the soil-water interface into the floodwater, intensive signals of CH4 oxidation were observed by following changes in isotopic signature between them (Fig. 7). An obvious oxidation signal was also observed of the dissolved CH4 approaching to soil surface , . Therefore, Fox-value was reasonably calculated based on δ13C-value of CH4 in pore water for δ13CH4 (initial) and on δ13C-value of CH4 in floodwater for δ13CH4 (final). It was high on D16 and D88, but relatively low on D47 and D50, especially in Treatment Flooding (Table 3). As the emission of CH4 in the fields goes absolutely through aerenchyma of the plants in the middle of the season, a very high percent of the CH4 is therefore consumed in the rhizosphere and a low percent oxidized at the soil-water interface (Table 3). On the contrary, CH4 emits into the atmosphere mainly through bubble ebullition and molecular diffusion in the early and the late rice-growing season, the CH4 is probably oxidized at the soil-water interface, showing a relatively high Fox-value as a consequence (Table 3). On the whole, there was no difference in mean Fox-value between the two treatments (47%) throughout the entire observational period, indicating that water management in the winter fallow season has little impact on CH4 oxidation at the soil-water interface. Notably, Krüger et al.  had even pointed out that porewater CH4 was a poor indicator of produced CH4. Therefore, CH4 in soil pore water on D47 and D50 in the present study might be oxidized partially as well. As a result, its δ13C-value was possibly not fit to stand for δ13CH4 (initial), which would bias the estimation of CH4 oxidation therein. Actually, the CH4 produced in paddy fields would be mostly oxidized in the rhizosphere because over 90% of the CH4 is considered to emit into the atmosphere through the aerenchyma of the plants while less than 0.1% released via ebullition and diffusion , , . Moreover, the absolute rates of CH4 oxidation at the soil-water interface were significantly lower than those in the rhizosphere –. Therefore, although the fraction of CH4 oxidized at the soil-water interface appears to be very high (Table 3), the amount of the CH4 must be significantly lower than that oxidized in the rhizosphere, and may be negligible.
In lab conditions, the difference between anaerobic and aerobic CH4 productions in the soil was apparently attributed to CH4 oxidation at the soil-water interface . In the present study, CH4 from aerobic incubation has undergone intensive oxidization, relative to CH4 produced anaerobically (Figs. 3d, e and 4a, b). This was reflected both in significantly low production rate (Fig. 5a, c) and more positive δ13C-value of the produced CH4 (Fig. 7). Therefore, the fraction of CH4 that was oxidized in aerobic condition could be estimated directly by using Eq. (8) based on δ13C-values of anaerobically produced CH4 for δ13CH4 (initial) (Figs. 3e and 4b) and on δ13C-values of aerobically produced CH4 for δ13CH4 (final) (Fig. 5b, d). Although little is known about the isotope fractionation when CH4 oxidation occurs on rice roots, αox = 1.038 was tentatively used to estimate Fox-value thereupon as well as in paddy soil. Results show that a variation pattern of Fox-value was similar to that in the rhizosphere, which was the highest in the first half of the season but tended to get lower in the second (Fig. 9). For soil, Fox-value ranged from ∼5 to 50% and was slightly affected by water management in the winter fallow season (Fig. 9c). For roots however, it was over 100% in the most of the season, suggesting that fresh rice roots consume almost all the produced CH4 by themselves in lab conditions. Moreover, it was 15% lower in Treatment Drainage than in Treatment Flooding (Fig. 9d). It is a matter of fact that little CH4 was produced in aerobic incubation and it even became negative in growth at the end of the season (Fig. 3c). In addition, drainage significantly decreased the CH4 oxidation capacity of the field relative to flooding (Fig. 6c). Methanotrophs are found to attach closely to, or even live inside, rice roots , , . The roots per se have a strong CH4 oxidation capacity indeed (Fig. 6c). On the other hand, it further indicates that value of αox = 1.038 may be unreasonable for roots in estimation of Fox, because in field conditions, CH4 production directly or indirectly from the roots must not be completely oxidized and Fox-value should be lower than 100%. Therefore, more investigation of fractionation factor αox in paddy soil, in particular on rice roots, should be performed to better quantify CH4 oxidation in the fields.
Through the field and laboratory experiments, we investigated δ13C in every process of CH4 emission from rice fields as affected by water management in the winter fallow season and further estimated pathways of CH4 production and fraction of CH4 oxidation using the stable carbon isotope technique. Compared with flooding, drainage generally caused the produced CH4 depleted in 13C. Although drainage significantly decreased CH4 emission, it had little effect on δ13C-value of emitted CH4, as well as the transport fractionation factor εtransport. Acetate-dependent methanogenesis dominated in the soil in the late season, but H2/CO2-dependent methanogenesis occurred mostly on the rice roots over the season. Drainage decreased the contribution of acetate to CH4 production by 5–10%. In field conditions, ∼10–90% of the CH4 was oxidized in the rhizosphere, while ∼30–70% at the soil-water interface. In lab conditions, less a half of the CH4 was oxidized in the soil, while almost all on the roots. Moreover, CH4 oxidation was more important in the first half of the season as well as in the rhizosphere. Drainage increased the fraction of CH4 oxidized in the rhizosphere by 5–15%, which is possibly attributed to the fact that CH4 production decreased significantly while CH4 oxidation did not simultaneously. Measuring δ13C-values of the CH4 from different pools in the rice fields is useful for quantifying the methanogenic pathway and the fraction of CH4 oxidized in these fields. More importantly, it is useful for better understanding the processes of CH4 emission, which may provide useful information for setting up an isotope model. Such a model may be of a great help to national or global CH4 budget. Therefore, more attentions should be paid to the paddy fields with more different patterns of agricultural management at a larger scale.
Conceived and designed the experiments: HX JM GZ. Performed the experiments: GZ JM GL YZ. Analyzed the data: HX GZ. Contributed reagents/materials/analysis tools: HX KY. Wrote the paper: GZ HX. Obtained permission for use: HX GZ.
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