CH₄ production potential in a paddy soil exposed to atmospheric CO₂ enrichment

Abstract

An anaerobic incubation experiment was conducted to investigate methane (CH₄) production potential in soil samples collected from a paddy field after exposure to free-air CO₂ enrichment (FACE). The FACE experiment with two CO₂ levels, ambient and ambient + 200 p.p.m.v CO₂ during the rice growing season, was conducted at Shizukuishi, Iwate Prefecture, Japan. The soil was a wet Andosol. Soil samples were taken from the surface (0–1 cm) and the sub-surface (1–10 cm) soil layers 2 months after rice harvest. Sub-samples of the fresh soils were put into glass bottles and submerged under N₂ gas headspace during the incubation. The results showed that, prior to incubation, the contents of total C and dissolved organic C (DOC) were significantly greater in FACE soil than ambient soil. During the incubation, CH₄ production potential was approximately 2–4-fold higher in FACE soil than ambient soil and approximately 500–1,000-fold greater in surface soil than sub-surface soil. In general, the FACE soil contained more DOC than ambient soil, particularly in the surface soil layer. These findings suggest that FACE treatment exerted long-term positive effects on CH₄ production and increased organic C content in this paddy soil, particularly in the surface soil layer.

Key words:

• dissolved organic C
• FACE
• methane
• paddy soil

INTRODUCTION

The atmospheric CO₂ concentration has been increasing at a rate of 0.5% annually and the major increase has occurred since the 1850s (Lal and Kimble 1995). Fossil fuel combustion and land-use changes are two principal human activities responsible for the increase. Increased atmospheric CO₂ can stimulate rice biomass production (above and belowground) and grain yield (Kim et al. 2003; Sakai et al. 2001; Ziska et al. 1997). Stimulated root growth can release more carbonaceous substances to soil in the form of rhizodeposition, such as root exudates, secretion, sloughed-off cells and decaying roots (Li and Yagi 2004; Lynch and Whipps 1990).

Dissolved organic C (DOC) is a mobile and labile form of soil organic C. In general, root exudates and microbially degraded products are regarded as the most important DOC sources. Elevated CO₂ can affect DOC dynamics in soils by increasing the amount of soluble organic C compounds from roots, and by stimulating or depressing decomposition of soil organic matter depending on N supply levels. However, most studies on elevated CO₂ effects have been carried out in upland soils (Cheng and Johnson 1998; Ginkel et al. 2000; Schortemeyer et al. 1996) and only a few studies have been conducted in paddy soils (Cheng et al. 2001; Hoque et al. 2001; Inubushi et al. 2003). The direct effect of elevated CO₂ on DOC dynamics would be expected to affect CH₄ production and emission from paddy soils indirectly, but such an effect has not been extensively identified. Furthermore, most investigations on CH₄ production and emission have been conducted during the rice growing season (Hoque et al. 2001; Inubushi et al. 2003), but limited information is available on CH₄ production potential in rice soils collected after rice harvest or during the off-crop period.

The objective of this research was to investigate CH₄ production potential in surface and sub-surface soil samples collected 2 months after rice harvest from a paddy field exposed to free-air CO₂ enrichment in Japan.

MATERIALS AND METHODS

The paddy soil used in this study was collected from a field experiment that was conducted under free-air CO₂ enrichment (FACE) conditions at Shizukuishi, Iwate Prefecture, Japan (latitude 39°40′, longitude 141°E, elevation 200 m a.s.l.). The soil was a wet Andosol, a typical paddy soil in Iwate Prefecture. The FACE experiment with two CO₂ levels, ambient and ambient + 200 p.p.m.v CO₂, was designed using a randomized complete block with four replicates. The FACE plots were named A, B, C and D, and the ambient plots were named W, X, Y and Z. The FACE facility was fully described by Okada et al. (2001).

Rice seedlings (Oryza sativa L cv. Akitakomachi) were raised in growth chambers for 3 weeks at Tohoku National Agricultural Experiment Station, Morioka, Japan. The seedlings for ambient CO₂ plots were grown in a chamber supplied with ambient CO₂ concentration, and those for FACE plots grown in a separate chamber with elevated CO₂ (ambient + 200 p.p.m.v). The seedlings were then transplanted by hand into the field at a density of three plants per hill and 19 hills m⁻² on 20 May 2004. Nitrogen fertilizers were applied as basal dressing at a rate of 80 kg N ha⁻¹, of which 60 kg was supplied as controlled release urea and 20 kg as ammonium sulfate. Phosphorus and K fertilizers were applied as basal dressings at a rate of 130 kg P ha^{−1 and} 120 kg K ha⁻¹, respectively. The above fertilizer rates (N–P–K) are close to the standard fertilizer rates (90–130–125 kg ha⁻¹ N–P–K) in the region of Japan (Hoque et al. 2001). Throughout the growing season (i.e. from transplanting until harvest), CO₂ was continuously supplied and released above the rice plant canopy, and diluted by wind turbulence in the FACE plots. Rice was

Table 1 Changes in root dry biomass (g m−2) in plots exposed to free-air CO2 enrichment (FACE) and ambient CO2

Treatment	12 July 2004 (Tillering)	21 September 2004 (Harvesting)
FACE	92.66 ± 5.45a	40.23 ± 3.85c
Ambient	80.31 ± 5.34b	30.33 ± 5.92d
Values are the mean ± standard error. Means in a column followed by the same letter were not significantly different using an independent samples t-test (P ≤ 0.05).

manually harvested on 21 September 2004 to avoid disturbing the soil layers by mechanical harvest. After harvest, straw was removed from the field and the stubble and roots were left in the field. To investigate root growth in response to CO₂ enrichment, root biomass was measured at tillering and harvest (Table 1). Root biomass was measured from two soil cores with hills at a depth of 0–30 cm from each plot (i.e. in total eight hills from ambient CO₂ plots and elevated CO₂ plots, respectively). Roots were washed with water, oven-dried at 70°C for 4 days and weighed.

Soil samples (surface 0–1 cm and sub-surface 1–10 cm) were separately collected on 2 December 2004 from FACE (plots B, C and D) and ambient (plots W, Y and Z) fields after rice harvest. Plots A and X had been ploughed before our sampling. After removal of all visible plant debris, roots and small stones, the soil was kept at 5°C in a refrigerator prior to incubation. Selected properties of the soil samples used in this experiment are listed in Table 2. Total C and N contents were determined using a CN analyzer (MT-700 with an Auto Sampler MTA-600; Yanaco, Kyoto, Japan). Soil pH value (1:1, H₂O) was measured using a pH meter (HORIBA/F-13, HORIBA Advanced Techno, Co., Ltd., Kyoto, Japan.).

After homogenization, 10 g sub-samples of the fresh soils (equivalent to 5 g oven-dried soil, in triplicate) were placed into glass bottles (50 mL) and submerged under a 2 cm water layer during the entire incubation. The air in the bottles was removed by evacuation and replaced with N₂ gas after the evacuated bottles were sealed with butyl rubber stoppers. The soils were incubated

Table 2 Selected properties of the soil samples used in this experiment

Treatment	Soil layer	Total C (g kg^−1)	Total N (g kg^−1)	DOC (µg C g^−1 dried soil)	pH (H2O, 1:1)
FACE	Surface	93.88 ± 1.50a	6.78 ± 0.06a	41.60 ± 0.92a	5.87 ± 0.11b
	Sub-surface	86.26 ± 1.21b	5.97 ± 0.07bc	29.44 ± 0.65b	5.91 ± 0.01b
Ambient	Surface	82.23 ± 1.44b	5.81 ± 0.08c	35.76 ± 0.79c	6.07 ± 0.10a
	Sub-surface	75.55 ± 1.30c	5.10 ± 0.05c	26.42 ± 0.59d	6.08 ± 0.07a
Values are the mean ± standard error. Means in a column followed by the same letter were not significantly different using a Tukey's test (P ≤ 0.05). DOC, dissolved organic carbon; FACE, free-air CO2 enrichment.

at 25°C in the dark. Gas samples were collected after 0, 2, 5, 10 and 15 days of incubation. Before gas sampling, the bottles were gently shaken by hand for 1 min to release the CH₄ dissolved in the submerged water layer to the headspace. After that, 0.5 mL of the gas samples in the headspace was collected and immediately injected into a gas chromatograph with Flame ionization detector (FID) (GC, Shimadzu 14B, Kyoto, Japan) to measure CH₄ concentration.

At the end of the incubation, the bottles were vigorously shaken by hand to obtain soil slurries. The slurries were transferred to centrifuge tubes, and centrifuged at 2,150 g for 15 min. The supernatants were filtered through a 0.40 µm Millipore filter and stored at −20°C. The organic C content in the solutions regarded as DOC was analyzed using a total carbon analyzer (TOC-5000, Shimadzu, Japan).

Statistical analysis was carried out using SPSS software (SPSS Inc., Chicago, Illinois, USA). All data were expressed as mean ± standard error and subjected to anova, Tukey's and t-tests (P < 0.05).

RESULTS

Root dry biomass and soil chemical properties

Root dry biomass was 10–12 g m⁻² more under FACE conditions than ambient conditions at each sampling date (Table 1). Compared with the ambient treatment, FACE treatment significantly increased total C and total N contents and DOC content in both the surface soil and sub-surface soil. Higher contents of total C and total N as well as DOC were obtained in the surface soil than the sub-surface soil. In contrast, pH values were markedly decreased in the soil samples with FACE compared with ambient CO₂ (Table 2).

CH₄ production potential in surface and sub-surface soils

In general, CH₄ production potential increased from the start to the end of the incubation (Fig. 1). No significant difference in CH₄ production potential was found between the FACE and ambient treatments at the early stage of the incubation, that is, within 2 days for surface soil and 10 days for sub-surface soil. However, towards the end of the incubation, CH₄ production potential became significantly higher in FACE soil (collected from FACE plots) than ambient soil (taken from ambient plots), regardless of soil depth. Furthermore, at the end of incubation a marked difference in CH₄ production potential was observed between surface soil and sub-surface soil, and CH₄ production potential was approximately 500–1,000-fold greater in surface soil than sub-surface soil.

Figure 1  Change in CH4 production from (a) surface (0–1 cm) and (b) sub-surface (1–10 cm) soil samples collected from a rice paddy field exposed to elevated and ambient CO2 concentrations. Vertical bars indicate the standard error of the mean. Values are the mean of three replicates. Asterisks indicate significant differences between free-air CO2 enrichment (FACE) and ambient treatments using an independent sample t-test (P ≤ 0.05).

Dissolved organic C content in surface and sub-surface soils

The DOC content was measured at the end of the incubation (Fig. 2). A significant difference in DOC content was observed in surface soil samples between the FACE and ambient treatments, but not in sub-surface soil samples. The amount of DOC obviously increased with FACE treatment in surface soil samples compared with the ambient treatment. For the FACE treatment, higher DOC content was detected in surface soil than sub-surface soil samples.

DISCUSSION

Free-air CO₂ enrichment obviously stimulated root biomass production (Table 1), which is similar to our

Figure 2  Dissolved organic C (DOC) in surface (0–1 cm) and sub-surface (1–10 cm) soil samples collected from a rice paddy field exposed to elevated and ambient CO2 concentrations. Columns with the same letter were not significantly different using Tukey's tests (P ≤ 0.05). Vertical bars indicate the standard error of the mean. Values are the mean of three replicates.

previous report (Inubushi et al. 2003), and increased total C and DOC contents in both surface and sub-surface soils. In addition, total C and DOC contents were markedly higher in surface soil than sub-surface soil, particularly under FACE conditions (Table 2). The above results implied that more plant-derived organic C (e.g. plant residues and debris) was allocated into the surface soil, as reported by some researchers (Li and Yagi 2004). Furthermore, our previous results showed that the amounts of chlorophyll-type compounds (an index of algal growth) and microbial biomass C and N were also higher in the surface soil than sub-surface soil under CO₂ elevation (Inubushi et al. 1999; 2001). Surface soil generally contained more easily decomposable organic matter (e.g. plant residues and algal debris) than sub-surface soil (Inubushi et al. 1982). The plant-derived C (mainly insoluble organic C) and chlorophyll-type compounds (algal debris) were initially decomposed to soluble or dissolved organic C, and further changed into CO₂ and CH₄. Thus, more plant residues and algal debris possibly returned to the FACE surface layers might be the reasons for the higher DOC content and greater CH₄ production potential in the FACE surface soil (Figs. 1,2).

During the rice growing period, methane production was enhanced by CO₂ enrichment because of the stimulated root biomass in the form of root exudates or root autolysis products, which were readily decomposed by fermentative bacteria to CO₂, H₂ and acetate, and then utilized by methanogens and further changed into CH₄ (Inubushi et al. 2003; Li et al. 2004; Minoda et al. 1996). In the above experiments, the soil samples for measuring CH₄ production were taken during the rice growing season. However, the soil samples tested in the present study were collected 2 months after rice harvest. The labile organic C, such as low molecular weight organic substances (e.g. organic acids and amino acids), contained in root exudates had possibly been degraded during the off-crop period. After rice harvest, plant-derived C remained in the soil at least in three forms: root residues, stubble and rhizodeposits (Kimura et al. 2004). An incubation experiment with ¹³C-tracer showed that, after 60 days of incubation, rhizodeposits C in water-soluble organic C decreased more rapidly than those in microbial biomass C and soil organic C, and further indicated that plant-derived C (root and stubble) accounted for 18–28% of total water-soluble organic C, 17–32% of total microbial biomass C and 2.3–4.1% of total soil organic C, respectively (Lu et al. 2003). In addition, during the rice growing season, microbial biomass C was enhanced in paddy soil, particularly in the surface soil layer of FACE treatment (Inubushi et al. 2001). Thus, the higher CH₄ production potential in the FACE surface soil (Fig. 1) might be caused by the decomposition of microbial biomass C and water-soluble organic C derived from photosynthesized C allocated to soil during the rice growing season (Hoque et al. 2001; Inubushi et al. 2001). Another possibility is that high molecular weight organic substances derived from the rice plant were decomposed to low molecular weight substances contributing to CH₄ production. Our results suggest that FACE treatment greatly affected C storage and turnover in the paddy soil, and exerted long-term effects on CH₄ production, particularly in the surface soil layer.

In conclusion, as the tested soil samples were collected 2 months after rice harvest, this study provided an insight into the long-term effects of FACE on CH₄ production potential, as well as C sequestration in the paddy soil. The FACE treatment exerted long-term positive effects on CH₄ production and increased the organic C content in the paddy soil, particularly in the surface soil layer. Further research is needed to investigate the contribution of surface soil to CH₄ production in the flooded paddy field under CO₂ enrichment.

ACKNOWLEDGMENTS

This study was financially supported by the Japan Society for the Promotion of Science in the form of a postdoctoral fellowship to Dr Y. Lou (P04429), the International Foundation for Science (C/3593) and the Rice-FACE project under the CREST (Core Research for Evolutional Science and Technology) program of Japan Science and Technology Corporation. We would like to express our special thanks to Dr Padre Agnes from the International Rice Research Institute, Philippines, for checking this manuscript.