Mitigate CH4 emission by suppressing methanogen activity in rice paddy soils using ethylenediaminetetraacetic acid (EDTA)

Geoderma 219–220 (2014) 58–62

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Mitigate CH4 emission by suppressing methanogen activity in rice paddy soils using ethylenediaminetetraacetic acid (EDTA) Prabhat Pramanik ⁎, Pil Joo Kim ⁎⁎ Division of Applied Life Science, Gyeongsang National University, Jinju 660701, South Korea

a r t i c l e

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Article history: Received 21 August 2013 Received in revised form 10 December 2013 Accepted 21 December 2013 Available online xxxx Keywords: Paddy soils Methane emission mitigation EDTA application Plant growth

a b s t r a c t Methane (CH4) is the second most potent greenhouse gases after carbon dioxide. More than 90% of world rice is cultivated under submerged condition, which facilitates CH4 production in soil. In this pot experiment, different doses of EDTA were applied in rice paddy soils to evaluate their effects on CH4 emission and plant growth during rice cultivation. Application of EDTA at small doses (up to 5.0 ppm) significantly (P b 0.05) suppressed CH4 emission without compromising rice grain yield. Higher doses (10.0 ppm) of EDTA application extended vegetative growth stage of rice plants, which not only reduced ripening percent of rice grains but also increased CH4 emission (even more than control). Therefore, based on this pot experiment data it could be concluded that EDTA application at 5.0 ppm was probably the most rational treatment to mitigate CH4 emission from rice paddy soils. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Methane (CH4) is considered as the second most potent greenhouse gas after carbon dioxide (CO2). It has 25 times higher global warming potential (IPCC, 2007) and 20% higher radiative forcing (IPCC, 2001) as compared to CO2. Approximately 1 billion tons of CH4 is globally formed per year by methanogens. Therefore, about 1.5% of the total 70 Gt of CO2, fixed annually into biomass by photosynthesis, is converted into CH4 (Thauer, 1998). Methanogenesis is an enzyme-mediated multi-step process by methanogens. Though methanogens may differ in terms of their preference for initial substrates (formate, acetate and/or CO2–H2); all of them synthesize a common compound namely coenzyme M (Co-M: 2-mercaptoethane sulfonate) (Ferry and Kastead, 2007; Grahame and Gencic, 2000). In the penultimate step, methylated Co-M is reduced by methyl Co-M reductase (MCR) enzyme to CH4 involving a nickel-containing cofactor F430 (Kaster et al., 2011). The activity of MCR enzyme is dependent on the F430 (Thauer et al., 2008) and therefore, the bioavailability of Ni to methanogens is expected to influence MCR activity and CH4 production in soil. Pramanik and Kim (2013a) revealed that incubation of soil with EDTA under anaerobic condition significantly decreased Ni bioavailability, reduced Co-M concentration and mcrA gene (responsible for synthesizing MCR enzyme) copy numbers in methanogens and hence suppressed CH4 production in soil. ⁎ Correspondence to: P. Pramanik, Soils Department, Tocklai Experimental Station, Tea Research Association, Jorhat 785008, Assam, India. Tel.: +91 87239 65361; fax: +91 376 2360 0974. ⁎⁎ Corresponding author. Tel.: +82 55 772 1966; fax: +82 55 772 1969. E-mail addresses: [email protected] (P. Pramanik), [email protected] (P.J. Kim). 0016-7061/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.geoderma.2013.12.024

Rice is one of the most staple foods in the world and is generally cultivated under flooded condition. Submerged rice paddy fields are recognized as an important source of CH4 emission (Xiong et al., 2006). The hypothesis of this study was that EDTA application might be effective to mitigate CH4 emission from rice paddy soils; however, its effect on soil nutrient status and rice plant growth was not known as it was the first attempt to use EDTA in rice paddy soil for mitigating CH4 emission. In this experiment, three different doses of EDTA were applied in rice paddy soil under greenhouse condition and changes in CH4 emission fluxes were correlated to the soil chemical and biochemical properties. The objective of this study was to evaluate the possibility of using EDTA for mitigating CH4 emission from rice paddy soils.

2. Materials and methods 2.1. Experiment setup The experiment was conducted in the greenhouse at agricultural field of Gyeongsang National University (36° 50′ N and 128° 26′E), Jinju, South Korea. The soil of this experiment was poorly drained with clay loam texture and was classified as andiaquands. Organic carbon (C) and nitrogen contents of initial soil were 11.79 ± 1.51 g kg−1 and 0.94 ± 0.09 g kg−1, respectively with C/N ratio of 12.54 and soil pH was 6.32 ± 0.43 (soil: water = 1:5, w/v). That soil was first homogenized, air-dried and 13 kg of that soil was packed in pots having surface area equal to 1/2000 acre land. Pots were then flooded with water and allowed to stand for stabilization (filling up of capillary pores with water). After stabilization of soil conditions, fertilizers and EDTA were applied and 3 rice (Dongjinbyeo cultivar, Japonica type)

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seedlings (25 days old) were transplanted in each pot. The chemical properties of initial soil were presented in Table 1. Chemical fertilizers were applied in each pot (including control) at N–P2O5–K2O = 1.12– 1.63–0.56 g pot−1 following the recommended doses (N–P2O5– K2O = 90–45–58 kg ha− 1) for Korean rice paddy soils. EDTA was applied at four doses, 0 ppm, 2.5 ppm, 5.0 ppm and 10.0 ppm (ppm amounts of EDTA were calculated considering the weight of pot soil). The ‘bases’ of cylindrical chambers were fixed in each pot and then the pots were arranged in the greenhouse following completely randomized design. Water management practices were also followed according to the recommendation for Korean rice paddy fields and levels of water in each pot were maintained at 5–7 cm above the soil surface. Gas samples, soil temperatures and Eh readings were collected after every 4 days, while soil samples were collected from each pot at 20 day intervals. After rice harvesting, above-ground portions were air-dried and grain and straw yields were recorded for all the treatments. 2.2. CH4 gas sampling and analysis A closed-chamber method (Ali et al., 2009) was used to estimate CH4 flux from soil for the entire cultivation period. Cylindrical acryl chambers having diameter 21 cm and height 100 cm were placed on ‘bases’ of each pot during gas sample collections. The gas samples were collected by using 50 ml air-tight syringes at 0, 15 and 30 min after placing the chambers to check the linearity and daily gas sampling was carried out at 0800, 1200 and 1600 h to find the optimum time for gas sample collection. Based on these data, gas samples were collected initially and 30 min after closing the chamber within 1030 and 1200 h at 4 day intervals throughout the rice cultivation period. Methane concentrations in the collected gas samples were measured by gas chromatography (Shimadzu, GC-2010, Japan) equipped with Porapak NQ column (Q 80–100 mesh) and a flame ionization detector (FID). The temperatures of column, injector and detector were adjusted at 100 °C, 200 °C, and 200 °C, respectively. Helium and H2 were used as carrier and burning gases, respectively. Methane emission from soil was calculated from the increase in CH4 concentrations per unit surface area of the chamber within a specific time interval. A closed-chamber equation (Rolston, 1986) was used to estimate CH4 fluxes from each treatment.

59

Total CH4 flux for the entire cultivation period was computed following the equation proposed by Singh et al. (1999): n

Total CH4 flux ¼ ∑i ðRi  Di Þ where Ri was the CH4 emission flux (g m−2 d−1) in the ith sampling interval, Di was the number of days in the ith sampling interval, and n was the number of sampling intervals. 2.3. Coenzyme M concentration in soil Coenzyme M (Co-M), an intermediate compound of methanogenesis, was quantified as a biomarker of methanogens in soil (Pramanik and Kim, 2012). The fresh soil was homogenized with lysis buffer (100 mM Tris–HCl solution (pH 8.0), 100 mM EDTA solution (pH 8.0) and 1.5 M NaCl solution) (Soil: buffer = 1: 2, w/v basis) and sonicated for 2 min (1 minute sonication followed by 10 second vertex and then 1 minute sonication again). The soil suspension was centrifuged at 4000 rpm for 10 min. Required amount of ethanol was added to the supernatant to make it 80% ethanol solution. The solution mixture was allowed to stand for 2 h at 4 °C and centrifuged again at 4000 rpm for 10 min. The precipitate was dissolved in deionized water and diluted to the suitable volume for HPLC analysis using UV detector at 270 nm (Pramanik and Kim, 2012). The mixture of acetonitrile and 0.05 M trichloroacetic acid solution (30:70, v/v) was used as mobile phase for Co-M quantification. 2.4. Microbial biomass C in soil Soil microbial biomass was estimated by the fumigation–extraction method of Vance et al. (1987). The fresh soil was fumigation with chloroform and C from fumigated (Cf) and non-fumigated (Cnf) soils were extracted by 0.5 M K2SO4 solution. The differences in these C contents give the measure of soil microbial biomass MBC ¼ ðC f –Cnf Þ=Kc where Kc, the correction factor, is equal to 0.45 for an agricultural soil (Wu et al., 1990).

F ¼ ρ  ðV=AÞ  ðΔc=ΔtÞ  ð273=TÞ

2.5. Methanogen activity in soil

where F was the CH4 flux (mg CH4 m−2 h−1), ρ was the gas density (0.714 mg cm−3), V was the volume of chamber (m3), ‘A’ was the surface area of chamber (m2), Δc/Δt was the rate of increase of CH4 gas concentration in the chamber (mg m− 3 h−1) and T (absolute temperature) was calculated as 273 + mean temperature in (°C) of the chamber.

To measure methanogen activity, fresh soil (10 g) was incubated with 1 ml of 1% glucose solution and 25 ml deionized water under anaerobic condition at 30 °C for 5 h (Pramanik and Kim, 2013b). The methanogen activity was measured by estimating CH4 concentration at the head-space of the bottles and the values were expressed as μg CH4 produced g−1 soil h−1.

Table 1 Soil nutrient status, rice grain yield and yield attributes as affected by different EDTA treatments. Categories

Parameters

Control

2.5 ppm EDTA

5.0 ppm EDTA

10.0 ppm EDTA

Soil chemical properties

Soil organic C (%) Total N (%) Nitrate–N (mg kg−1) Available P2O5 (mg kg−1) Exchangeable K2O (mg kg−1) Grain yield (t ha−1) Straw yield (t ha−1) Harvest index Plant height (cm) Tiller numbers Number of grains panicle−1 1000 grain weight Ripening percent

1.206 ± 0.273 0.071 ± 0.005 16.94 26.5 ± 3.6 247.2 ± 31.6 1.80 ± 0.09 2.98 ± 0.22 0.61 95.8 ± 3.3 24.3 ± 1.5 124.5 ± 39.1 22.22 ± 1.19 82.1 ± 3.9

1.086 ±0.174 0.071 ± 0006 15.38 32.1 ± 2.8 264.0 ± 33.7 1.91 ± 0.12 2.94 ± 0.06 0.65 101.0 ± 1.0 26.7 ± 1.5 113.1 ± 29.0 22.77 ± 0.61 85.2 ± 1.8

1.288 ± 0.227 0.074 ± 0.003 17.43 29.5 ± 3.1 255.2 ± 42.8 2.06 ± 0.08 2.63 ± 0.29 0.79 100.0 ± 3.5 27.3 ± 3.1 140.9 ± 40.2 22.07 ± 1.92 86.7 ± 0.7

1.183 ± 0.325 0.069 ± 0.004 17.25 31.4 ± 3.2 230.9 ± 26.7 1.56 ± 0.12 1.68 ± 0.20 0.75 87.7 ± 5.3 22.7 ± 1.2 99.2 ± 28.3 21.96 ± 1.59 74.1 ± 2.6

Rice yield

Yield attributes

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2.6. Soil chemical analysis Soil organic carbon (SOC) content was measured by following the standard dichromate oxidation method of Nelson and Sommers (1982). Total N and exchangeable K contents in soil were determined by the standard methods described by Jackson (1973). For measuring available P contents, soil samples were extracted with 0.5 N sodium bicarbonate solution and blue color in the aliquot was developed by ammonium molybdate solution under acidic condition. The blue color intensity was compared with those of standard solutions to measure available P content in soil (Olsen and Sommers, 1982). To determine DOC content, fresh soil samples were homogenized with deionized water (soil:water = 1: 5, w/v basis) by shaking at 120 rpm for 1 h (Lu et al., 2011). After extraction, the suspension was centrifuged at 4000 rpm for 15 min and the supernatant was used for organic carbon determination by Shimadzu total organic carbon analyzer (model 5000A). Soil was extracted with 2 M KCl solution (soil:extractant = 1:10, w/v basis) and the filtrate was then vortexed after adding 1 ml of 30% NaCl solution and 5 ml of 4:1 sulfuric acid solution. The solution was then cooled down and 0.25 ml of coloring reagent (1 g brucine and 0.1 g sulfonic acid was dissolved in 100 ml of 3% HCl solution). The solution mixture was then warmed in water-bath at 100 °C for 20 min, cooled down and vortexed well to read the absorbance at 410 nm (Jackson, 1973). The sample readings were then compared with that of standard curve to determine NO3–N content in soil samples. 2.7. Statistical analyses Statistical analyses were conducted using SAS software (SAS Institute, 1995). Rice yield, soil properties, and methane emission data were subjected to the analysis of variance and regression. Fisher's protected least significant difference was calculated at the 0.05 probability level for comparing treatment means. 3. Results 3.1. CH4 emissions from rice paddy soils Methane was emitted from rice paddy soils throughout the rice cultivation period, though the rates of CH4 emission were depending on the period of rice cultivation (Fig. 1). Irrespective of treatments, the highest CH4 emission fluxes were observed within 20–50 days after seedling transplanting. The highest CH4 emission flux was recorded

Control 2.5 ppm EDTA 5.0 ppm EDTA 10.0 ppm EDTA

6

4

2 20

Coenzyme M concentrations in soil were varied depending on the applied treatments and cultivation period. Irrespective of the time of rice cultivation, the highest Co-M concentrations were observed in control soil and EDTA application significantly (P b 0.05) reduced Co-M concentration in soil (Fig. 3a). Initially, the Co-M concentrations of 2.5 ppm and 5.0 ppm EDTA treated soils were comparatively higher than that of 10.0 ppm EDTA treated soils, which was significantly (P b 0.05) increased after 50 days of transplanting. During this latter stage of cultivation, the Co-M concentrations of 10.0 ppm EDTA treated soils were even higher than those of control soils. 3.3. Microbial biomass C (MBC) Microbial biomass C in rice paddy soils was also varied depending on the time of rice cultivation and EDTA doses (Fig. 3b). Rice cultivation steadily increased MBC in soil up to 60 days after transplanting, and MBC values in control soil were gradually decreased thereafter. EDTA application decreased MBC of soil and recorded comparatively lower MBC than control soil up to 40 days of rice cultivation. However, MBC of all EDTA treated soils after 60 days of rice cultivation was statistically at par with that of control soil and unlike control soil, MBC of EDTA treated soils was slightly increased thereafter. At the latter stage of rice cultivation (after 80 days), MBC of EDTA treated soils was significantly (P b 0.05) higher than that of control soil. 3.4. Methanogen activity Rice cultivation increased methanogen activity in soil up to 60 days after transplanting (Fig. 3c). Methanogen activity was steadily decreased thereafter and methanogen activity during harvesting was almost similar as that of initial soils. EDTA application significantly

130

8

0

3.2. Co-M concentrations in soil

Cumulative CH4 emission (kg ha-1)

CH4 emission flux (mg m-2 h-1)

10

in control treatment and EDTA application reduced the rate of CH4 emission from soil. Initially, the rates of CH4 emissions were inversely proportional to the doses of EDTA application; however, 10.0 ppm EDTA application recorded comparatively higher rates of CH4 emissions (sometimes even higher than control, too) at the latter stages of rice cultivation. The cumulative CH4 emission during rice cultivation was 118.28 ± 4.97 kg ha−1 from control soil. EDTA application significantly (P b 0.05) reduced the values of cumulative CH4 emission, though the CH4 emissions from different EDTA treated soils did not vary significantly among them (Fig. 2). The lowest CH4 emission was observed from 5.0 ppm EDTA treated soils (96.84 ± 3.68 kg ha−1).

40

60

80

100

Days after transplanting Fig. 1. CH4 emission fluxes from rice paddy soils as affected by different EDTA treatments.

120

110

100

90 10 0 Control

2.5 ppm EDTA

5.0 ppm EDTA 10.0 ppm EDTA

Treatments Fig. 2. Cumulative CH4 emission from different rice paddy soils.

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Co-M concentration (µmol g-1)

400

a

350

3.6. Rice grain yields and plant growth parameters Control 2.5 ppm EDTA 5.0 ppm EDTA 10.0 ppm EDTA

Grain and straw yields of rice were varied due to EDTA application at different doses (Table 1). The grain yield of control treatment (1.80 ± 0.09 t ha−1) was increased up to 1.91 ± 0.12 t ha− 1 and 2.06 ± 0.08 t ha−1 as a result of 2.5 ppm and 5.0 ppm EDTA application, while 10.0 ppm EDTA treatment recorded lower grain yield as compared to control. The highest yield index (0.79) was recorded in 5.0 ppm EDTA treatment. Generally, EDTA application up to 5.0 ppm improved quality of rice grains as compared to control. However, mostly this change was not significant (Table 1). EDTA application at 10.0 ppm significantly (P b 0.05) reduced the ripening percent and hence deteriorated the quality of rice grains.

300

250

200

150

Methanogen activity (µg CH4-C g-1 hr-1)

Microbial biomass C (µg g-1)

80

b

60

4. Discussion

Control 2.5 ppm EDTA 5.0 ppm EDTA 10.0 ppm EDTA

40

20

0 2.5

c

2.0

61

Control 2.5 ppm EDTA 5.0 ppm EDTA 10.0 ppm EDTA

1.5

1.0

0.5

0.0 20

40

60

80

100

Days after transplanting Fig. 3. Changes in coenzyme M (Co-M) concentrations (a), microbial biomass C (b) and methanogen activity (c) in rice paddy soils as affected by different EDTA applications.

(P b 0.05) reduced methanogen activity in soil and recorded lower methanogen activity up to 80 days of rice cultivation as compared to control soil. After 80 days of transplanting, EDTA application at 5.0 ppm or less recorded similar methanogen activity as that of control, though higher doses of EDTA application (at 10.0 ppm) had shown higher methanogen activity in rice paddy soils. 3.5. Soil nutrient status Soil organic C and total N contents were not changed significantly due to EDTA application in rice paddy soils (Table 1). However, NO3–N contents in EDTA treated soils were higher than that of control. Nitrate–N contents of 5.0 ppm and 10.0 ppm EDTA treated soils were significantly (P b 0.05) higher than that of control. The highest NO3–N content was recorded in 10.0 ppm EDTA treated soils. Available P2O5 and exchangeable K contents in soil did not show any relation with the rates of EDTA application (Table 1).

Rice is generally cultivated under submerged field condition, especially in Asian countries (Crutzen, 1995; Yu and Patrick, 2004). Continuous flooding shifted soil redox to the reduced condition (Supplementary Fig. 1), which favors the methanogenesis in soil (Garica et al., 2000; Takai, 1961). Methanogens convert simple organic C compounds into CH4 through enzyme-mediated multi-step methanogenesis process (van den Pol-van Dasselaar and Oenema, 1999). Pramanik and Kim (2013a) revealed that EDTA application limited Ni availability to methanogens and that in turn reduced CH4 emission from soil. They proposed that available Ni2+ ion in EDTA-treated soil combined with this chelating compound and that significantly reduced its bioavailability to methanogens. Limited bioavailability of Ni2+ ions suppressed the activity of MCR enzyme in methanogens and which in turn reduced the rate of methanogenesis in soil. However, the effect of EDTA application on methanogenesis in rice paddy soil was not known. The pot experiment suggested that EDTA application in the abovementioned rates significantly (P b 0.05) mitigated CH4 emission from rice paddy soils; however, it also affected rice plant growth and the activity of other soil microorganisms. Interactions of EDTA with MBC or rice plant growth were varied depending on the rates of EDTA applications. Concentrations of Co-M were determined as the biomarkers of methanogens (Pramanik and Kim, 2012). EDTA application at 2.5 ppm also decreased Co-M concentration and mcrA gene copy numbers in soil; however, the maximum effect of EDTA application on methanogens was observed when 5.0 ppm or more EDTA was applied to the pot soils. The changes in microbial activity were calculated as substrate-induced microbial respiration (SIR) using the formula of Pramanik and Kim (2013b): MBC ¼ kcf ð1:956 MA þ 0:710 SIR þ 10:192Þ; where kcf ¼ 2:142  0:337 i:e: SIR ¼ 0:658 MBC–14:355 MA–2:755 where MBC was microbial biomass C, MA was methanogen activity and kcf was correction factor. Initially, EDTA application suppressed SIR along with methanogen activity in soil (Supplementary Fig. 2) and the extent of decrease was proportional to the doses of EDTA applications. Thereafter, SIR in soil was gradually increased and after 20–30 days of rice cultivation, these treatments recorded comparatively higher SIR values as compared to control. The lowest SIR was recorded due to 10.0 ppm EDTA application and reached the initial SIR levels only after 40–45 days of rice cultivation. Based on these microbiological data, it could be concluded that EDTA application at 5.0 ppm or less could be the effective treatment to suppress methanogen activity without reducing activities of other microorganisms in rice paddy soils. Based on soil chemical and microbiological properties, rice grain yields were also varied depending on the doses of EDTA applications. Plants were visibly different due to EDTA applications in rice paddy

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soils (Supplementary Fig. 3). Low doses of EDTA applications increased rice grain yield and the highest grain yield in 5.0 ppm EDTA treated pots might be attributed to the improved soil chemical and biological properties in these soils. The lowest microbial respiration was possibly responsible for the suppressed plant growth in 10.0 ppm EDTA treated soils (low SPAD readings; Supplementary Fig. 4). Though SIR in this treatment was gradually increased with time, the high SIR was achieved after 60 days of rice cultivation. Though the highest NO3–N content at the harvesting stage was recorded in 10.0 ppm EDTA-treated soils, comparing with SIR data it could be concluded that soil NO3–N content was probably increased at the latter stage of rice cultivation. Higher NO3–N content after 40–50 days of transplanting extended the vegetative stage and delayed the reproductive stage (Masclaux-Daubresse et al., 2010) of rice plants which was indicated by the highest SPAD readings even after 80 days of rice cultivation and low ripening percent of rice grains. Therefore, it could be concluded that high EDTA doses (above 5.0 ppm) had a detrimental effect on soil properties and rice plant growth, though it also suppressed methanogen activity in rice paddy soils. Hence, according to the findings of this pot experiment, EDTA application at 5.0 ppm is possibly the most rational dose to mitigate CH4 emission from soil without compromising chemical and microbiological properties of soil and rice plant growth. 5. Conclusion EDTA application reduced CH4 emission by suppressing methanogen activity in rice paddy soils. High doses of EDTA application deteriorated soil chemical and microbiological properties. 5.0 ppm EDTA treatment significantly suppressed methanogen activity and CH4 emission without compromising soil chemical and biochemical properties and rice productivity. Based on the findings of this pot experiment, 5.0 ppm EDTA application is possibly the rational dose to mitigate CH4 emission from rice paddy soils. Acknowledgement This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC grant: PJ009087), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.geoderma.2013.12.024. References Ali, M.A., Lee, C.H., Lee, Y.B., Kim, P.J., 2009. Silicate fertilization in no-tillage rice farming for mitigation of methane emission and increasing rice productivity. Agric. Ecosyst. Environ. 132, 16–22.

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