Carbon isotope fractionation reveals distinct process of CH₄ emission from different compartments of paddy ecosystem

Abstract

Carbon isotopic fractionations in the processes of CH₄ emission from paddy field remain poorly understood. The δ¹³C-values of CH₄ in association with production, oxidation and transport of CH₄ in different pools of a paddy field were determined, and the stable carbon isotope fractionations were calibrated to assess relative contribution of acetate to CH₄ production (f_ac) and fraction of CH₄ oxidized (f_ox) by different pathways. The apparent isotope fractionation for CO₂ conversion to CH₄ (α_app) was 1.041–1.056 in the soil and 1.046–1.080 on the roots, indicating that f_ac was 10–60% and 0–50%, respectively. Isotope fractionation associated with CH₄ oxidation (α_ox) was 1.021 ± 0.007 in the soil and 1.013 ± 0.005 on the roots, and the transport fractionation (ε_transport) by rice plants was estimated to be −16.7‰ ~ −11.1‰. Rhizospheric f_ox was about 30–100%, and it was more important at the beginning but decreased fast towards the end of season. Large value of f_ox was also observed at the soil-water interface and soil and roots surfaces, respectively. The results demonstrate that carbon isotopic fractionations which might be different in different conditions were sensitive to the estimations of f_ac and f_ox in paddy field.

Methane (CH₄) is the second most important greenhouse gas after carbon dioxide (CO₂). On a 100-year horizon, CH₄ has 25 times the global warming potential of CO₂. Paddy fields are one of the largest anthropogenic sources of atmospheric CH₄, contributing to 33–40 Tg yr⁻¹ during the 2000–2009¹. The global CH₄ emission from paddy fields will continually increase by intensification of rice cultivation and expansion of planting area to meet the demands of the growing populations²,³,⁴. Paddy CH₄ emission is an integrated effect of the production, oxidation and transport of CH₄ in the field. A better knowledge of these processes affecting CH₄ emission may provide more information for effectively mitigating CH₄ emission in agricultural ecosystems.

The technique of stable carbon isotopes has been proved to be a useful tool in studying the processes of CH₄ emission⁵,⁶,⁷,⁸,⁹. Isotope fractionation happens in all the major processes CH₄ emission, namely, ¹²C-substrate is preferentially utilized by methanogens for CH₄ production, and once formed, ¹²CH₄ is consumed faster than ¹³CH₄ by methanotrophs, and ¹²CH₄ is transported faster than ¹³CH₄ as well¹⁰,¹¹,¹². Thereby, measurements of the δ¹³C in production, oxidation and transport of the CH₄ from different pools of the field are benefical to support a process-based model for CH₄ emission⁸,¹³,¹⁴. Moreover, the relative contribution of acetate to CH₄ production (f_ac) and the fraction of CH₄ oxidized (f_ox) can be quantitatively estimated⁵,⁶,⁷ using mass balance equations based on the measurements of δ¹³C in CH₄, CO₂ and acetate, and of the isotope fractionation factors (, α_ox and ε_transport). Investigations on , and ε_transport of paddy fields were carried out greatly⁵,¹⁵,¹⁶,¹⁷,¹⁸, however, few data are available on α_ox for CH₄ oxidation by methanotrophs in paddy soils, in particular α_ox on rice roots¹⁹.

Some uncertainties also exist in the δ¹³CH₄ that are used as newly produced δ¹³CH₄ (δ¹³CH_{4 (original)}) and finally oxidized δ¹³CH₄ (δ¹³CH_{4 (oxidized)}) in different studies for quantifing f_ac and f_ox. For example, former reports in USA using porewater δ¹³CH₄ as δ¹³CH_{4 (original)}⁶,⁷ whereas anaerobically produced δ¹³CH₄ was used in Italy⁵,²⁰ and China¹⁹,²¹. They believed that porewater CH₄ was a poor proxy for δ¹³CH_{4 (original)} as it was potentially affected by CH₄ oxidization and transport in field conditions¹¹,²². Similarly, various δ¹³CH₄, such as rhizospheric δ¹³CH₄, aerobically produced δ¹³CH₄, porewater δ¹³CH₄ or floodwater δ¹³CH₄ sometimes, have been regarded as δ¹³CH_{4 (oxidized)} for estimation of f_ox in the rhizosphere or at the soil-water interface⁵,⁶,⁷,⁹,¹⁹,²³. More importantly, large differences were observed in the estimated f_ac and f_ox if different δ¹³CH_{4 (original)} and δ¹³CH_{4 (oxidized)} was assumed in the same study⁵,⁷. Therefore, more comparable observations in different conditions with corresponding isotope fractionation factors are needed to discuss δ¹³CH_{4 (original)} and δ¹³CH_{4 (oxidized)} in accurate estimations of f_ac and f_ox.

In this study, field and incubation experiments were conducted to observe the process of CH₄ emission closely related to the production, oxidation and transport of CH₄, including CH₄ fluxes emitted from the field and via the plants, CH₄ concentrations in the aerenchyma of the plants, and in soil pore water and floodwater, CH₄ production and oxidation rates in the soil and on the roots, and all the corresponding δ¹³CH₄. The objectives of the present study were (1) to improve our understanding of the processes in CH₄ emission by measurements of the stable carbon isotopes, (2) to investigate the isotope fractionation factor α_ox in the soil and on the roots, and (3) to discuss the availabilities in the estimation of f_ac and f_ox associated with different pools of δ¹³CH₄ in the field.

Results

CH₄ production and δ¹³C of CH₄ and CO₂

In anaerobic incubation, both CH₄ production potentials in the soil and on the roots were relatively low on 20 days after rice transplanting (D20), peaked (2.2 μg CH₄ g soil⁻¹ d⁻¹ and 11.1 μg CH₄ g root⁻¹ d⁻¹) on D50, and then turned downwards gradually to the bottom on D108 (Table 1). For δ¹³C-value of the produced CH₄, it was more and more positive in the soil during the whole observational period, being from −71.1‰ to −53.9‰ (Table 1). For CH₄ produced on the roots however, it was most ¹³C-depleted on D50 and then ¹³C-enriched again on D88, with δ¹³C-value ranging between −86.9‰ and −66.6‰ (Table 1). Throughout the whole season, δ¹³C-value of produced CO₂ increased from −18.8‰ to −14.9‰ in the soil while decreased from −15.0‰ to −23.3‰ on the roots (Table 1). CH₄ production under aerobic incubation was hardly observed in the soil (0.06 to 0.13 μg CH₄ g soil⁻¹ d⁻¹), particularly on the roots, which was lower than 0 μg CH₄ g root⁻¹ d⁻¹ over the whole season (Fig. 1a). The δ¹³C-value of CH₄ produced in the soil was relatively stable around −55‰ while on the roots it was about −40‰ (Fig. 1b). Apparently, the δ¹³C-values of CH₄ produced in aerobic incubation were significantly higher (P < 0.05) than those of the CH₄ that was produced in anaerobic incubation (Table 1 and Fig. 1).

Table 1. CH₄ production potentials (μg CH₄ g⁻¹ d⁻¹), δ¹³C-values (‰) of CH₄ and CO₂ in the soil and on the roots under anaerobic incubation, and the corresponding apparent fractionation (α_app) between CO₂ and CH₄ calculated by the ratio of (δ¹³CO₂ + 1000)/(δ¹³CH₄ + 1000).

Days after rice transplanting (d)	Production		δ13CH4		δ13CO2		αapp
	Soil	Root	Soil	Root	Soil	Root	Soil	Root
20	0.13 ± 0.18	3.4 ± 0.7	−71.1 ± 2.4	−69.4 ± 2.8	−18.8 ± 2.9	−15.0 ± 2.3	1.056 ± 0.005	1.058 ± 0.002
50	2.15 ± 0.21	11.1 ± 2.2	−64.4 ± 0.4	−86.9 ± 3.5	−17.0 ± 1.8	−14.1 ± 1.7	1.051 ± 0.002	1.080 ± 0.003
88	0.38 ± 0.12	4.5 ± 0.6	−57.5 ± 1.1	−66.6 ± 2.7	−15.1 ± 0.5	−24.0 ± 2.9	1.045 ± 0.001	1.046 ± 0.005
108	0.22 ± 0.03	3.2 ± 0.9	−53.9 ± 0.2	−72.5 ± 2.9	−14.9 ± 0.4	−23.3 ± 2.8	1.041 ± 0.000	1.053 ± 0.001

Figure 1.

Temporal variations of CH₄ production rates in the soil and on the roots under aerobic (a,b) incubation, and corresponding δ¹³CH₄.

CH₄ concentration and δ¹³C of CH₄ and CO₂

CH₄ concentration in soil pore water was more than 100 μM L⁻¹ in most part of the season (Fig. 2), and it was highest (~120 μM L⁻¹) on D50. CH₄ concentration in floodwater was in the range of 0.21–2.6 μM L⁻¹, being significantly lower than that of soil pore water over the season (P < 0.01). The δ¹³C-values of CH₄ in soil pore water and floodwater appeared to increase simultaneously (Fig. 2), from ~−70‰ to −60‰ and from ~−50‰ to −40‰, respectively. Obviously, CH₄ in soil pore water was more depleted in ¹³C than that of floodwater CH₄ (P < 0.05), indicating that porewater CH₄ was intensively affected by CH₄ oxidation at the soil-water interface when it released into the atmosphere. CO₂ in soil pore water tended to ¹³C-enriched gradually during the rice season, with δ¹³C-values ranging from −20.0‰ to −14.5‰ (Fig. 2).

Figure 2. Temporal variations of CH₄ concentrations in soil pore water and floodwater, and corresponding δ¹³C-values of CH₄ and CO₂.

CH₄ oxidation and δ¹³C of CH₄

CH₄ oxidation potential in the soil peaked on D50 (6.9 μg CH₄ g soil⁻¹ d⁻¹), and then it dropped gradually to the lowest on D108 (Table 2). In contrast, it was highest on the roots (580 μg CH₄ g root⁻¹ d⁻¹) on D20 and decreased sharply on D50. After an increase on D88, it decreased again to the lowest on D108 (Table 2). The δ¹³C-values of CH₄ before oxidization were −41.0‰ ~ −38.4‰ in the soil and −40.5‰ ~ −36.0‰ on the roots. After CH₄ oxidization, the CH₄ both in the soil and on the roots were more enriched in ¹³C, with δ¹³C-values of −38.4‰ ~ −32.5‰ and −34.0‰ ~ −26.5‰, respectively (Table 2).

Table 2. CH₄ oxidation potentials (μg CH₄ g⁻¹ d⁻¹), δ¹³C-values (‰) of CH₄ at time 0 (δ¹³CH_{4 (initial)}) and at time t (δ¹³CH_{4 (final)}) in the soil and on the roots under aerobic incubation with high CH₄ concentration supplemented, and the corresponding CH₄ oxidation fractionation factor (α_ox) calculated by the Equation (5).

Days after rice transplanting (d)	Oxidation		δ13CH4 (initial)		δ13CH4 (final)		αox
	Soil	Root	Soil	Root	Soil	Root	Soil	Root
20	4.4 ± 1.4	580 ± 116	−38.4 ± 1.6	−38.7 ± 1.9	−35.6 ± 2.2	−34.0 ± 2.8	1.014 ± 0.002	1.019 ± 0.005
50	6.9 ± 1.1	335 ± 84	−41.0 ± 0.4	−40.4 ± 0.7	−35.0 ± 1.7	−26.5 ± 1.9	1.020 ± 0.002	1.012 ± 0.007
88	5.1 ± 1.9	454 ± 68	−38.7 ± 1.1	−40.5 ± 2.6	−32.5 ± 1.0	−30.6 ± 2.2	1.030 ± 0.004	1.015 ± 0.003
108	2.3 ± 1.3	258 ± 78	−40.3 ± 0.0	−36.0 ± 0.2	−38.4 ± 0.2	−31.2 ± 3.4	1.021 ± 0.009	1.008 ± 0.009

Plants emitted and aerenchymatic CH₄

On the three sampling days (D37, D62 and D98) during the season, CH₄ emitted via the plants was relatively stable with δ¹³C-values of −63.9‰, −62.6‰ and −63.5‰, respectively. For δ¹³C-values of aerenchymatic CH₄, they were −49.2‰, −45.9‰ and −52.4‰, respectively, being significantly higher in comparison of the emitted CH₄ (P < 0.05). As a result, the isotope fractionations due to CH₄ transport (ε_transport) were measured to be −14.7‰, −16.7‰ and −11.1‰, respectively, with a mean value of −14.2‰.

CH₄ emission and δ¹³C of CH₄

The CH₄ flux varied significantly, with the highest value appeared on D50 and the lowest on D108, ranging from 0.4 to 11.5 mg CH₄ m⁻² h⁻¹ during the observational period (Fig. 3a). The δ¹³CH_{4 (emission)} varied between −68.7‰ and −61.5‰ with the variation pattern just opposite to that of CH₄ flux (Fig. 3a). It is noteworthy that a significant negative relationship between CH₄ flux and corresponding δ¹³CH_{4 (emission)} was observed (Fig. 3b). Soil temperature ranged from 17.2 °C to 30.5 °C during the rice season, with a value of 24.5 °C on average.

Figure 3.

Temporal variations of CH₄ flux and δ¹³CH₄ (a) and they relationship (b).

δ¹³C of organic carbon in soil and plant samples

The values of δ¹³C in soil organic carbon did not show much variation during the rice season, being −26.84‰ on D37 and −27.66‰ on D108, respectively. The organic carbon in the plant samples also remained relatively stable over the season, with δ¹³C-values being −29.19‰ on D37 and −28.70‰ on D108, respectively, although they were slightly lighter than those of the soil organic carbon.

Discussion

CH₄ production

The processes of CH₄ production, oxidation, transport and emission from paddy field were well presented by the measurements of stable carbon isotopes in CH₄ from different pools of the field (Fig. 4). The decomposition of plants debris and root exudates, besides soil organic matters in the bulk soil, is very important to methanogenesis in paddy field²⁴. As a key precursor for methanogens, it was slight ¹³C-depletion on the roots relative to soil organic carbon (Fig. 4). Previous studies also showed δ¹³C-value of organic carbon relatively negative in the plant than in the soil⁵,¹⁹. Paddy field CH₄ is mainly produced out of either cleavage of acetate (f_ac) or reduction of H₂/CO₂ (1 − f_ac), and the δ¹³C-value of produced CH₄ primarily depends on relative contribution of the two main methanogenic pathways¹⁰,²⁵. The f_ac was calculated by the following mass balance⁶,⁷:

Figure 4. Stable carbon isotopes in the processes of CH₄ emission from the paddy field.

Note: each δ¹³C-value was given in arithmetic mean of the rice season.

During the process of acetate fermentation forming CH₄, isotopic fractionation occurs and the fractionation factor is generally expressed to . It was found to be −21‰ in pure cultures of acetoclastic Methanosarcina barkeri²⁶ and −18‰ for acetoclastic Methanosaeta concilii¹⁵,²⁷. Using ‰, Krüger et al.⁵ estimated δ¹³C of CH₄ produced from acetate (δ¹³CH_{4 (acetate)}) between −43‰ and −37‰ according to the measurements of δ¹³C_acetate (−22‰ ~ −16‰) in soil pore water of an Italian rice field. Meanwhile, both values of −43‰ and −37‰ have well been applied in many studies⁶,⁷,⁹,¹⁶,¹⁹,²¹. Due to a lack of knowledge on and in order to compare the data interpretation with those of above mentioned, both δ¹³CH_{4 (acetate)} of −43‰ and −37‰ were used in the present study (Table 3).

Table 3. Relative contribution of acetate to total CH₄ production (%) in the soil ( f _ac ^a) and on the roots ( f _ac ^b).

Days after rice transplanting (d)	faca				facb
	δ13CH4 (acetate) = −37‰		δ13CH4 (acetate) = −43‰		δ13CH4 (acetate) = −37‰		δ13CH4 (acetate) = −43‰
	= 1.050	= 1.060	= 1.050	= 1.060	= 1.070	= 1.080	= 1.070	= 1.080
20	−21 ± 4	8 ± 3	−27 ± 4	9 ± 3	24 ± 6	37 ± 7	28 ± 7	41 ± 8
50	−2 ± 6	23 ± 3	−3 ± 7	28 ± 3	−20 ± 1	0 ± 0	−23 ± 2	1 ± 0
88	18 ± 3	39 ± 2	24 ± 4	48 ± 3	42 ± 2	50 ± 15	47 ± 2	56 ± 17
108	32 ± 0	50 ± 0	42 ± 0	61 ± 0	29 ± 0	39 ± 11	33 ± 0	44 ± 12

f_ac^a and f_ac^b was calculated with Equation (2) using δ¹³C-values of CH₄ anaerobically produced in the soil and on the roots (Table 1) as originally produced δ¹³CH₄, respectively.

When H₂/CO₂ reduction produces CH₄, isotopic fractionation factor is defined by Hayes²⁸:

where is δ¹³C of the CH₄ produced from H₂/CO₂ reduction. In addition, based on the ratio of δ¹³CO₂ to δ¹³CH₄ in anaerobic incubation (Table 1), an approximation of apparent fractionation (α_app) between CO₂ and CH₄ can be calculated by using α_app = (δ¹³CO₂ + 1000)/(δ¹³CH₄ + 1000). The α_app is calculated from the isotopic signatures of total CH₄ produced from H₂/CO₂ reduction and acetate cleavage, and theoretically, it is lower than . Results of 16 different lake sediments from tropical freshwater wetlands in Brazil²⁹ have well demonstrated that (1.075 ± 0.008) is much higher than α_app (1.059 ± 0.009). In this study (Table 1), the α_app decreased gradually from 1.056 on D20 to 1.041 on D108 for the soil. In contrast, it increased sharply from 1.058 on D20 to 1.080 on D50, and then decreased again to 1.053 for the roots. Totally, α_app was relatively lower in the soil (1.041–1.056) than on the roots (1.046–1.080). The was hence assumed to be 1.050–1.060 in the soil and 1.070–1.080 on the roots (Table 3). Incubating three different soils, Conrad et al.¹⁷ also found that was between 1.050 and 1.060 for two paddy soils. Additionally, previous studies approved the relatively larger (≥1.070) on the roots than in the soil due to their methanogenic populations were different¹⁶,³⁰.

The CH₄ produced in anaerobic incubation changed significantly during the rice season, and it was much more ¹³C-enriched in the soil than on the roots (Table 1). It indicates that methanogenic pathway was changed with rice growing, and also demonstrates that acetate-dependant methanogenesis was more important in the soil. In this study, f_ac in the soil was initially very low (<10%) on D20, but it increased obviously with the rice growing and reached over 60% on D108. In contrast, f_ac on the roots was relatively high (~30–40%) on D20. It decreased sharply in the middle of the season (near 0%) and then increased again to about 50% on D108. As a whole, f_ac was relatively higher in the soil than that on the roots (Table 3). Previous study in Italian paddy soil has also demonstrated that acetoclastic methanogenesis was higher than 60% at the end of the season⁵. High contribution of H₂/CO₂-dependent methanogenesis to total CH₄ production on rice roots was considerably reported⁵,⁹,¹⁶,¹⁹, and the major reasons were supposed to be the methanogens population on rice roots dominated by Rice Cluster I archaea³¹,³²,³³. Methanogenic substrates of organic carbon in the plant appeared to be slightly ¹³C-depleted relative to those of the bulk soil (Fig. 4), which might be a potential reason for the lower f_ac in the soil.

On the other hand, Belik et al. and Tyler et al.⁶,⁷ estimated f_ac of the USA paddy fields by using porewater δ¹³CH₄ as δ¹³C-value of the produced CH₄, and they found that it was as high as 80% when . However, Canadian field data have showed that porewater CH₄ is possibly influenced by CH₄ oxidation and transport²², and in an Italian paddy field Krüger et al.⁵ also considered that porewater CH₄ was a poor proxy for produced CH₄ due to the potential CH₄ oxidation therein. Recently, a pot experiment in Germany suggested that porewater CH₄ could be used as newly produced CH₄ after tillering stage since they were similar in δ¹³C³⁴. In this study, both porewater δ¹³CH₄ and produced δ¹³CH₄ generally tended to be enhanced during the rice season (Table 1 and Fig. 2), and on average they were similar with each other (Fig. 4). According to δ¹³C-values of porewater CH₄ and CO₂ (Fig. 2), it was found that the α_app in soil pore water was from 1.047 to 1.054. Therefore, was assumed for comparing with former reports⁶,⁷ and present data of the paddy soil. Hydrogenotrophic methanogenesis was estimated to be dominated over the season (~60–80%), which differed much from the field data in USA⁶,⁷. More importantly, the methanogenic pathway in soil pore water was different from that in paddy soil (Table 3). Although reasons for the difference in f_ac between paddy soil and porewater were not clear, it is not recommended here that porewater CH₄ was absolutely regarded as newly produced CH₄.

CH₄ oxidation

The produced CH₄ in paddy field is mainly oxidized in the rhizosphere and at the soil-water interface, and accurate estimation of the CH₄ oxidation is one of the major aims of this study. Compared to δ¹³C of newly produced CH₄ (δ¹³CH_{4 (original)}), δ¹³C of remaining CH₄ after it has undergone oxidization (δ¹³CH_{4 (oxidized)}) was significant ¹³C-enriched (Fig. 4). Therefore, fraction of the CH₄ that is oxidized (f_ox) in the field can be estimated by the mass balance equation⁶,⁷:

In general, anaerobically produced δ¹³CH₄ is regarded as δ¹³CH_{4 (original)} and δ¹³CH_{4 (oxidized)} is estimated by the measurements of δ¹³CH_{4 (emission)} corrected with transport fractionation factor (ε_transport) using a semi-empirical equation⁵,⁷,¹⁶:

In the closed-system incubation, CH₄ oxidation fractionation factor α_ox is known to be calculated according to the Rayleigh equation³⁵,³⁶:

where δ¹³CH_{4 (initial)} stands for δ¹³C-value of CH₄ at time 0, δ¹³CH_{4 (final)} for δ¹³C-value of CH₄ at time t, and f (%) for the percentage of CH₄ remaining at time t.

To our knowledge, α_ox = 1.025 − 1.038 at a temperature of 12–35 °C is initially measured in methanotrophs-enriched cultures³⁵ and then widely in landfill cover soils³⁶,³⁷,³⁸, and it has substantially been used in the studies of paddy soil⁵,⁶,⁷,⁹,²⁰,²¹,²³. Recently, α_ox = 1.025 − 1.033 was found in a Chinese paddy soil at 28.3 °C¹⁹. By far, reports on α_ox in paddy soil, in particular on rice roots, are very few available. In the present study (Table 2), α_ox in the soil firstly increased from 1.014 on D20 to the highest value of 1.030 on D88, and then it decreased again to 1.021 on D108. In contrast, α_ox on the roots generally declined from 1.019 on D20 to the lowest 1.008 on D108. As a whole, it was higher in the soil (1.021 ± 0.007) than on the roots (1.013 ± 0.005) at 24.5 °C, being much lower than those of measured or used in previous studies under a similar temperature as above mentioned. In addition to α_ox-value of 1.021 ± 0.007 in the soil and 1.013 ± 0.005 on the roots was used, we made an alternative calculation using α_ox = 1.038 for better comparable to the previous studies (Table 4). Reasons for the difference in α_ox between paddy soils and rice roots are not understood, but Jahnke et al.³⁹ found that there were complex factors influencing the isotopic fractionation in CH₄ oxidation and carbon assimilation in various methanotrophs. Besides, main groups of methanotrophs in rice microcosm (Methylococcaceae and Methylocystaceae) are active, but their dominance may change depending upon substrate supply and nutrient status⁴⁰,⁴¹. Therefore, it is no wonder that our measurement of α_ox in paddy soil was different from that on rice roots, and that both of them differed much from that found in different environments and habitats as above mentioned. On the other hand, ε_transport is equivalent to the difference between emitted and aerenchymatic δ¹³CH₄⁵,⁷, and it was estimated to be −14.2‰ on average (Detailed descriptions please see below).

Table 4. Fraction of CH₄ oxidized (%) in the rhizosphere ( f_ox^a) and at the soil-water interface ( f_ox^b) in field conditions, and at the surfaces of soil ( f_ox^c) and rice roots ( f_ox^d) in lab conditions.

Days after rice transplanting (d)	αox = 1.021			αox = 1.013	αox = 1.038
	foxa	foxb	foxc	foxd	foxa	foxb	foxc	foxd
20	108 ± 16	88 ± 16	78 ± 18	235 ± 26	61 ± 9	49 ± 7	44 ± 10	82 ± 9
50	51 ± 6	116 ± 25	61 ± 6	372 ± 43	29 ± 3	65 ± 14	34 ± 3	130 ± 16
88	42 ± 16	86 ± 22	27 ± 3	209 ± 27	23 ± 9	48 ± 12	15 ± 2	73 ± 10
108	33 ± 22	84 ± 12	−4 ± 13	244 ± 24	19 ± 12	47 ± 7	−2 ± 8	86 ± 9

f_ox^a was calculated with Equation (5) using δ¹³C-values of CH₄ anaerobically produced in the soil (Table 1) as δ¹³CH_{4 (original)} and δ¹³CH_{4 (emission)} (Fig. 3a) minus −14.2‰ as δ¹³CH_{4 (oxidized)};

f_ox^b was calculated with Equation (5) using δ¹³C-values of CH₄ in soil pore water (Fig. 2) as δ¹³CH_{4 (original)}, and δ¹³C-values of CH₄ in floodwater (Fig. 2) as δ¹³CH_{4 (oxidized)};

f_ox^c and f_ox^d were calculated with Equation (5) using δ¹³C-values of CH₄ anaerobically produced in the soil and on the roots (Table 1) as δ¹³CH_{4 (original)} and δ¹³C-values of CH₄ aerobically produced in the soil and on the roots (Fig. 1) as δ¹³CH_{4 (oxidized)}, respectively.

Rhizospheric CH₄ oxidation was the most important on D20 (Table 4). At that time, almost the produced CH₄ was oxidized before it was emitted into the atmosphere. With the rice growing, the f_ox decreased fast to ~30% in the end. Both CH₄ oxidation potentials in the soil and on the roots were highest between D20 and D50, and decreased gradually towards the end of the season (Table 2), which might be the important reason. In situ inhibitor experiments, Krüger et al.⁴² also found that f_ox was highest just at the beginning of the season with a peak of ~40%, and then it became negligible at the end of the season. Soon later, it was reported that f_ox was no more than 50% over the season and it decreased rapidly from the beginning till the end of the season⁵,²⁰. They further concluded the possible reason was that activities of the methanotrophs were limited by nitrogen consumption with the rice growing under field conditions⁵,²⁰,⁴².

When porewater CH₄ released into the floodwater of the paddy fields, it was strongly oxidized at the soil-water interface since floodwater CH₄ was much more ¹³C-enriched than porewater CH₄ (Fig. 2). So, f_ox in this oxidizing area, in principle, can be estimated using porewater δ¹³CH₄ as δ¹³CH_{4 (original)} and floodwater δ¹³CH₄ as δ¹³CH_{4 (oxidized)}. Value of f_ox was found to be over 80% throughout the whole season, which was generally higher than that of f_ox in the rhizosphere (Table 4). Although f_ox at the soil-water interface appeared to be much high, the amount of the CH₄ oxidized must be significantly lower than that in the rhizosphere. Because produced CH₄ is mostly oxidized in the rhizosphere during the rice-growing season as over 90% of the CH₄ emits to the atmosphere through the aerenchyma of the plants while less than 0.1% releases via ebullition and diffusion²⁴,⁴³,⁴⁴. In addition, it was reported that the absolute CH₄ oxidation rate at the soil-water interface was much lower than that in the rhizosphere²⁴,⁴⁵,⁴⁶.

Compared to methanogenesis in anaerobic soil, that was in aerobic soil significantly lower in CH₄ production rate but more positive in δ¹³C (Fig. 4). The findings demonstrate that intensive CH₄ oxidization happened at the soil surface in lab conditions. As a result, f_ox at the soil surface (Table 4) was estimated using anaerobically produced δ¹³CH₄ as δ¹³CH_{4 (original)} and aerobically produced δ¹³CH₄ as δ¹³CH_{4 (oxidized)}¹⁹. It was the highest (~80%) at the beginning of the season and decreased rapidly later (<0%). In field conditions, CH₄ oxidation in paddy field without rice plants occurs mainly at the soil-water interface, which is similar to CH₄ oxidation under aerobic incubation in lab conditions. Therefore, it is feasible to quantitatively estimate f_ox in paddy fields during the non-rice-growing season or at the soil-water interface during the rice-growing season based on the difference in δ¹³CH₄ between anaerobic and aerobic incubations. What is more, f_ox at the root surface was also estimated by comparing δ¹³C-value of the CH₄ produced under aerobic conditions with those under anaerobic conditions (Table 4). It was found that f_ox at the root surface stayed over 100% throughout the whole season. Even if the α_ox = 1.038 was used, it was still as high as 100% (Table 4), further suggesting that CH₄ oxidation on rice roots was extremely strong indeed. CH₄ oxidation rate much higher on the roots (Table 2) was supposed to be the main reason for the f_ox was higher than that in the soil.

CH₄ transport and emission

Transporting CH₄ is the last step of CH₄ emission from paddy field to the atmosphere. Although CH₄ oxidation leads to the produced CH₄ obviously enriched in ¹³C, isotope fractionation in CH₄ transport offsets the positive effect on δ¹³CH₄, causing the CH₄ ¹³C-depleted again¹³,²². As a result, the δ¹³C-values of emitted CH₄ were close to the produced δ¹³CH₄ (Fig. 4). The isotope fractionation changes with the efficiency of CH₄ transport in growth of the plants⁵,⁹,²³. In the middle of the season, CH₄ transport capacity of the plants should get to highest because of full-developed rice plants and roots. Transport fractionation at that time is believed to be strongest and a value of −16.7‰ for ε_transport was measured on D62. At the beginning of the season or aging in the late part of the season, transport fractionation would be relatively weak due to the undeveloped plants with low CH₄ transport capacity. Therefore, the ε_transport was found to be −14.7‰ on D37 and −11.1‰ on D98. Many reports have shown a similar variation and it is generally between −16‰ and −11‰⁵,⁶,⁷,⁹,¹³,¹⁹.

Similar to CH₄ emission from paddy fields, δ¹³CH_{4 (emission)} is significantly affected by all each process of CH₄ production, oxidation and transport. At the beginning of the season, the high δ¹³CH_{4 (emission)} was most likely ascribed to the relatively low transport fractionation and the highest f_ox. Subsequently, it became lowest, which was supposed to be the biggest transport fractionation and significantly decreased f_ox. At the late period of the season, the emitted CH₄ was ¹³C-enriched again, mainly due to an obvious increase of f_ac at this moment. The δ¹³CH_{4 (emission)} was negatively correlated with CH₄ emission (Fig. 3b), which further indicates that the higher the CH₄ flux, the lower the f_ox, thus causing the lower the emitted δ¹³CH₄. Similar relationships were also observed in the previous studies⁹,²¹,⁴⁷.

In conclusion, this study well showed each process of CH₄ emission by the measurements of δ¹³CH₄ from various pools of the paddy field, and found that stable carbon isotope fractionation occurred in CH₄ production, oxidation and transport. Compared to the roots (1.046–1.080 and 1.013 ± 0.005), α_app was lower (1.041–1.056) whereas α_ox was greater (1.021 ± 0.007) in the soil. This suggests that acetate-dependent methanogenesis was more important in paddy soil whereas CH₄ oxidation was much stronger on the roots. Rice plant-mediated CH₄ transport fractionation (ε_transport) was found to be −16.7‰ ~ −11.1‰. Temporal variation of CH₄ emission negatively correlated with δ¹³CH_{4 (emission)} indicates the important relationships of CH₄ emission with production, oxidation and transport of the CH₄, which could be demonstrated by the changes of pathway of CH₄ production and fraction of CH₄ oxidation. Besides related newly produced δ¹³CH₄ and finally oxidized δ¹³CH₄, available carbon fractionation factors were needed to estimate relative contribution of acetate to total CH₄ production and fraction of CH₄ oxidized.

Methods

Experimental site

The experimental plots are located at Baitu Town, Jurong City, Jiangsu Province, China (31°58′N, 119°18′E). Soil of the field is classified as Typic Haplaquepts, with 11.1 g kg⁻¹ in total C, 1.3 g kg⁻¹ in total N and −26.8‰ in δ¹³C-value of soil carbon. After wheat was harvested on June 13, 2009, wheat straw with stubble and even wild weeds were all removed from the plots. Then the plots were kept flooded from June 24 to October 15 and drained on October 16 before rice harvest. Seeds of the rice (“Oryza sativa L. Huajing 3”) crop were sown into the nursery bed on May 25, seedlings were transplanted into the field on June 26, and the crop was harvested on November 3. Urea was applied at a rate of 300 kg N ha⁻¹, 50% as basal fertilizer on June 26, 25% as tillering fertilizer on July 17, and 25% as panicle fertilizer on August 16. Ca(H₂PO₄)₂ (450 kg ha⁻¹) and KCL (225 kg ha⁻¹) was applied with urea just as basal fertilizer on June 26.

Field sampling

CH₄ flux was monitored using the static chamber technique. The flux chambers (0.5 × 0.5 × 1 m), made of plexiglass, covered six hills of rice plants each. Plastic bases for the chambers were installed before rice transplantation in the plots. Removable wooden boardwalks (2 m long) were set up at the beginning of the rice season to avoid soil disturbance during sampling and measuring. To measure CH₄ flux, gas samples were usually collected once every 4–7 days. Four gas samples from each chamber were collected using 18 mL vacuum vials at 15 min intervals between 09:45 and 10:30 in the morning on each sampling day. To determine carbon isotope composition of the CH₄ gas (δ¹³CH_{4 (emission)}), samples were taken at 15–30 day intervals. Only two gas samples were collected using 0.5 L bags (aluminium foil compound membrane, Delin gas packing Co., Ltd, Dalian, China) with a small battery-driven pump⁴⁷. The first sample was taken after the chamber was closed for 3–5 min, and the second at the end of the 2 h closure period. When CH₄ flux was monitored, soil temperature at 10 cm depth was simultaneously measured with a hand-carried digital thermometer (Yokogawa Meter and Instruments Corporation, Japan).

Soil pore water samples, 10 cm in depth, were collected using a Rhizon soil moisture sampler (10 RHIZON SMSMOM, Eijkelkamp Agrisearch Equipment, Giesbeek, Netherlands)⁴⁷. The samplers were installed (in triplicate) prior to rice transplanting and then left in the soil throughout the whole season. Samples (about 5 mL) were firstly extracted using 18 mL vacuum vials to flush and purge the sampler before sampling. Then about 10 mL of soil solution was drawn into another vial. Simultaneously, 10 mL of floodwater was collected using a plastic syringe and then transferred in to an 18 mL vacuum vial. Subsequently, all sampling vials were equilibrated by filling in pure N₂ gas for further analysis with a GC-FID.

CH₄ emitted via rice plants and the aerenchymatic CH₄ was measured using specially designed PVC bottomless pots¹⁹. 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 adjustable in diameter to fit the growing plant in the center was placed on top of the pot, allowing the plant to grow through the hole and keeping the plant into two parts. Then, the plant in one pot was cut right above the plate while the plant in the other pot remained intact as control. Finally, chambers (0.3 × 0.3 × 1 m) were laid on the pots, and gas samples in the headspace of the chambers were collected simultaneously with a small battery-driven pump.

Soil cores of the top layer (0–15 cm) were collected at about 15–30-day intervals, and samples of the same plot were first mixed together⁴⁸. Two samples from the mixture, about 50 g each (dry weight), were then taken and transferred promptly into two 250-mL Erlenmeyer flasks separately. Samples in the flasks were prepared into slurries with N₂-flushed de-ionized sterile water at a soil/water ratio of 1:1. During the whole process, N₂ was constantly flushed through the samples to remove O₂ and CH₄. One flask was sealed for anaerobic incubation. Other flask with air headspace was sealed directly for aerobic incubation. Simultaneously, rice plants together with roots were carefully collected from the plots⁴⁸. The roots were washed clean with N₂-flushed demineralized water and cut off at 1–2 cm from the root with a razor blade. The fresh roots, about 20 g each portion, were put into flasks, separately, for further preparation and processing in the same way as for the soil samples. All the flasks were sealed with rubber stoppers fitted with silicon septum that allowed sampling of headspace gas. Finally, they were stored under N₂ at 4 °C and transported back to the lab as soon as possible for further analysis. A small portion of the soil and plant samples were dried for 72 h at 60 °C for determination of isotopic composition of the organic carbon.

Lab incubations

CH₄ production potentials were measured 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 incubated in darkness at a temperature the same as measured in the field for 50 h. Gas samples were analyzed 1 h and 50 h later after heavily shaking the flasks by hand. CH₄ production rates were calculated using the linear regression of CH₄ increasing with the incubation time.

CH₄ oxidation potentials were determined under aerobic incubation with high concentration of CH₄ supplemented, using equipment the same as described above. Firstly, pure CH₄ was injected into each flask to make a high concentration inside (~10,000 μL L⁻¹). Then, the flasks were incubated in darkness 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 analysis. The first sample was collected generally 30 min after pure CH₄ was injected. Samples were then taken at 2–3 h intervals during the first 8 h of the experiment. The flasks were left over night and sampled the next day at 2 h intervals again. CH₄ oxidation rates were calculated by linear regression of CH₄ depletion with incubation time.

Chemical measurements

CH₄ concentrations were analyzed with a gas chromatograph (Shimadzu GC-12A, Kyoto, Japan) equipped with a flame ionization detector. For analysis of carbon isotope composition, the continuous flow technique and a Finnigan MAT 253 isotope ratio mass spectrometer was used (Thermo Finnigan, Bremen, Germany)⁴⁷,⁴⁹. CO₂ in gas samples was directly analyzed while CH₄ in gas samples was converted into CO₂ and separated primarily on a PreCon (pre-concentration device). Then, the gas was piped into a GC equipped with a Pora PLOT Q column (25 m length; 0.35 mm i.d.) at 25 °C under 2.0 × 10⁵ Pa for further separation. The separated gases were finally transferred into the mass spectrometer for δ¹³C determination. The reference and carrier gases used were CO₂ (99.999% in purity and −23.73‰ in δ¹³C_PDB-value) and He (99.999% in purity, 20 mL min⁻¹), respectively. The precision of the repeated analysis was ±0.2‰ when 2.02 μL L⁻¹ CH₄ was injected. 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).

Statistical analyses

Statistical analysis was done using the SPSS 18.0 software for Windows (SPSS Inc., Chicago). Differences between the four treatments were determined through one-way analysis of variance (ANOVA) and least significant difference (LSD) test. Relationships were assessed using correlation analysis. Significant differences and correlations were set at P < 0.05.

Additional Information

How to cite this article: Zhang, G. et al. Carbon isotope fractionation reveals distinct process of CH₄ emission from different compartments of paddy ecosystem. Sci. Rep. 6, 27065; doi: 10.1038/srep27065 (2016).