Impact of Aquatic Earthworms on Methane Emission Reduction from the Paddy Field Soil in Japan

Methane (CH4) is one of the major greenhouse gases that significantly contributes to global warming. Therefore, substantial efforts are being made to reduce CH4 emissions. Paddy fields make a major contribution to atmospheric CH4 concentration because of their anoxic soil environment. Paddy field is habitat of many aquatic earthworms which can play a crucial role in reducing CH4 emissions from paddy field, because their bioturbation activities influence the soil structure and increase oxygen penetration and hence the activity of methanotrophs. Therefore, it was hypothesized that aquatic earthworms may accelerate oxidation of CH4 through their bioturbation activity. This incubation study evaluated the effects of earthworms on the activity of methanotrophs. Soil was incubated with a full factorial combination of two levels of aquatic earthworm (with and without earthworm) and two levels of fertilizer (with and without urea) for 35 days. The addition of urea increased the earthworm density in paddy soil by nearly doubled of without urea. At 28 days after incubation (DAI) the CH4 flux decreased from 2055 mg m h in only urea received soil to 425 mg m h in soil received both urea and earthworm treatment. Phospholipid fatty acid analysis showed that the presence of aquatic earthworms contributed to an increase in the biomass of methanotrophs. The study implies that aquatic earthworms may play vital role to reduce CH4 emission form paddy fields by creating favorable environment for methanotrophs, even in the soils fertilized with urea.


Introduction
The Paris Agreement has set a target to limit global warming to well below 2 °C (UNFCCC, 2015).Diversified efforts and actions to reduce emissions of major greenhouse gases (GHGs) are critical to achieve this ambitious, but important target.Methane (CH 4 ) is one of the most potent GHGs as its contribution to the greenhouse effect is almost half of that of carbon dioxide (CO 2 ), even though the atmospheric concentration of CH 4 is 1.8 ppm, lower than that of CO 2 (399 ppm) (Gavin, 2004;Blasing, 2016).CH 4 has been recognized as the second most important GHG after CO 2 , because of its higher global warming potential, which is 25 times higher than CO 2 (Shindell et al., 2009).A recent report showed, over the last 200 years, the atmospheric CH 4 concentration increased to double (Blasing, 2016).Wetland paddy fields significantly contribute to CH 4 emission, which is equivalent to 11% of total emission (Smith et al., 2007).
Rice is the staple food of more than half of the world's population, which is mainly grown in flooded field condition (Kögel-Knabner et al., 2010).Continuous flooding condition for over the rice cultivation period leads to the anoxic soil environment, which favors growth of unique microbial communities (Yao et al., 1999;Lüdemann et al., 2000;Kögel-Knabner et al., 2010).In absence of oxygen, biodegradation of organic matter results in the methanogenesis, which is the main terminal microbiological process in anoxic environment (Thauer, 1998).
The major methanogenic substrates are molecular hydrogen (H 2 ), CO 2 , formate, acetate, methyl alcohol, and methylamine; the dominant reactions of methanogenesis in soil are the reduction of CO 2 using molecular H 2 and the transmethylation of acetate (Takai, 1970).During methanogenesis, carbon substrates are supplied to soil from soil organic matter, sloughed tissues of rice plants, applied organic matter, and root exudates (Watanabe et al., 1999;Kimura et al., 2004).Methanotrophs consume some of the CH 4 produced under oxidative conditions in the rhizosphere of rice plants and in the thin layer where the soil and surface water meet (Dubey, 2005).The rest of the produced CH 4 is released from paddy fields into the atmosphere via diffusion, ebullition from soil, and by diffusion and mass flow through the continuous intercellular gas space system between the rice rhizosphere and leaves.Inubushi et al. (1989) reported that 90% of total CH 4 from paddy field is released through rice plant and ebullition and diffusion from soil contributes 10% and < 1%, respectively.The physiological structure of rice plants favors greater emissions of CH 4 .A path of gaseous exchange exists between the root zone and atmosphere; intercellular channels through the aerenchyma connect the rhizosphere to the leaves, where gases are released into the atmosphere via stomata in the leaves and micropores in the leaf sheaths.In submerged paddy fields, this allows the diffusion of CH 4 from the roots, to higher parts of the plant and then into the atmosphere (Nouchi et al., 1990).
Above discussions revealed that methanogenesis is the main process of CH 4 formation in the wetland rice field and dropping of oxygen supply is one of the main factors to stimulate this process.There are many management measures including change in tillage practice, modify irrigation and drainage practice, change of nutrient supply sources has been tested and found some potential measures to improve oxidation status of soil environment of wetland paddy field.For example, a single mid-season drainage results in significant reduction of CH 4 emissions (Kimura et al., 1992;Minamikawa et al., 2005).However, this management system increases emission risk of N 2 O (Bronson et al., 1997;Chen et al., 1997;Suratno et al., 1998).In a study, Yagi and Minami (1990) reported that chemical fertilizers strongly affect CH 4 emissions from paddy field soils.Urea is the most widely used nitrogenous fertilizer for growing wetland rice because it is easy to handle and reasonably priced (Jones et al., 2013).In 1961, worldwide consumption of urea was 2 million tons, whereas in 1998, worldwide consumption of urea was 86 million tons (Soh, 2001).Use of huge amount of urea for growing rice may have impact on CH 4 emission from paddy fields.Number of studies has been conducted to evaluate urea's impact on CH 4 emission in paddy fields.However, the effects of urea on CH 4 emissions from soils were found to be context-dependent.In some studies, CH 4 emissions increased with increasing urea application rates because of increasing soil pH and ammonium inhibited the growth of methanotrophs (Wang et al., 1992;Dubey et al., 2003).In contrast, other study reported that ammonium enhanced oxidation of CH 4 and the activities of methanotrophs (Bodelier et al., 2000).Aquatic earthworms are a major group of invertebrate fauna in paddy field ecosystems and maintain soil quality (Simpson et al., 1993).More specifically, aquatic earthworms in paddy soils improve soil health by stimulating the release of NH 4 + -N from the surface waters to the soils (Kikuchi & Kurihara, 1977, 1982;Fukuhara et al., 1980;Grant & Seegers, 1985) as well as PO 4 3- (Kikuchi & Kurihara, 1982).The earthworm gut and fresh cast of earthworms accelerate activity of methanogens (Depkat-Jakob et al., 2012).On the other hand, burrows made by earthworms may aerate the soil and stimulate methanotrophic activity (Kernecker et al., 2015).The bioturbation activity of aquatic earthworms affects microorganism community by enhancing oxidation (Yingst & Rhoads, 1980).Thus, aquatic earthworms may mitigate CH 4 emissions from flooded paddy soils through inhibiting methanogenesis process and stimulating growth of CH 4 -consuming methanotrophs, a biological sink of CH 4 .However, there has been no study conducted to examine the effect of aquatic earthworms in CH 4 emission from wet land paddy fields.It is also important to consider effect of fertilizer application on earthworms.However, low concentration of urea was reported as positive and beneficial for earthworm growth (Xiao et al., 2004).
Considering the above-mentioned background, we hypothesized that controlled application of urea and bioturbation activity by aquatic earthworms would enhance the biomass of methanotrophs, ultimately leading to a decrease in CH 4 emissions.To test this hypothesis, we designed a research study with a full factorial combination of two aquatic earthworm levels (with earthworm and no earthworm) and two levels of fertilizer (with urea and no urea).As of our knowledge, this is the first study, which examined potential role of aquatic earthworms in CH 4 mitigation process.The main objectives of our study were as follows: (i) To investigate effect of urea application on abundance of aquatic earthworms and CH 4 emission.
(ii) To see effect of aquatic earthworms on methanotrophs and CH 4 emission from wetland paddy soil.

Soil Preparation
The laboratory experiment was conducted in Soil Ecology Laboratory of Yokohama National University, Japan.A 2X2 factorial experiment with 6 replications was designed involving two levels in the aquatic earthworm treatment: the presence or absence of aquatic earthworms (EW and NW, respectively), and two levels of urea treatment (UR and NU, respectively).Soil was collected from a paddy field in Kamakura, Kanagawa (35°20′N, 139°31′E; 56 m.a.s.l.).The soil contained 40% sand, 58% silt, and 2% clay.The soil was sun-dried, plant debris and stones were removed, and then the soil was sieved using 2 mm sieve to achieve a homogenous texture.A 100 g of soil was added in each of 24 transparent glass bottles.Then 120 ml water was poured on soil in each bottle.

Collection of Aquatic Earthworms
Aquatic earthworms (Branchiura sowerbyi Beddard, 1892, Clitellata, Tubificidae) were collected from the flooded paddy soil, because it is one of the most dominant aquatic earthworm species in the paddy field (Yachi et al., 2012).The paddy soil was flooded for 8 weeks before collecting aquatic earthworms.To collect aquatic earthworms, muddy soils were sieved by a 500-µm mesh sieve.The earthworms were kept in deionized water for 48 h to excrete intestinal residues.Then, the live earthworms were placed on filter paper to remove extra water from the body and each earthworm was weighed to record their biomass.Collected aquatic earthworms were incubated in soil for 7 days.

Experimental Set-Up
Rice straw powder (2500 kg ha -1 ) was added to soil in each bottle as a source of organic matter and mixed gently using a spoon.As fertilizer treatment, granular urea (46% N) was added in respective 12 bottles at a rate of 90 kg N ha -1 .Pre-incubated 6 earthworms were inoculated in six urea treated bottles and six no-urea treated bottles.Number of inoculated aquatic earthworms was decided based on representative density of earthworms in the Kamakura paddy field (unpublished data), which was 2525 earthworms m -2 .
All bottles were incubated at 25 °C temperature and under 14 hours photoperiod to provide a nearly natural lighting environment for the earthworms for 35 days.At the end of the incubation period, the aquatic earthworms were collected from the soil in the bottles by sieving through a 500-µm mesh size sieve and the numbers and biomass of earthworms were recorded.

Gas Sampling and CH 4 Measurement
Gas samples were collected once per week, starting from day 0, during the experiment.The first sample was collected 2 h after preparing the soil and the bottles were placed in the sampling area 30 min before sampling.Then, the mouth of each bottle was closed with a rubber septum that had plastic lids running from it (to collect the gas produced).The first gas sample was collected 10 min after closing a bottle, and then after 20 and 30 min, with a plastic disposable 50 ml syringe.Gas samples were collected into 30-ml glass vials.Methane concentration was measured using a gas chromatograph (GC-2014, SHIMADZU, Japan) and CH 4 flux was calculated using the flux package (Jurasinski et al., 2014) in R software (R Development Core Team, 2015).

Monitoring Soil Properties
Each week soil pH and soil oxidation reduction potential (Eh) were measured at 1 cm and 5 cm depth from surface immediately after gas sample collections.Soil pH and Eh were measured using pH/COND Meter D-54 instrument (Horiba, Kyoto, Japan) and a pH/ORP/DO Meter D-75 instrument (Horiba, Kyoto, Japan), respectively.

Measuring Soil Properties at the End of Incubation Period
After collection of the final gas sample, soil samples were collected to measure total carbon (T-C) and total nitrogen (T-N).Collected samples were oven-dried at 40 °C for 48 h, and then T-C and T-N were measured using a CN Macro Corder (JM1000CN, J-Science Lab.Kyoto, Japan).

Phospholipid Fatty Acid (PLFA) Analysis
A phospholipid fatty acid (PLFA) analysis was conducted to determine the biomass of methanotrophs in this experiment.Lipids were extracted using a method modified from Frostegård et al. (1991), andNiwa et al. (2008).Briefly, lipids were extracted from 7-8 g fresh wet soil samples in a one-phase chloroform-methanol-phosphate buffer.After condensation of the lipids, the phospholipids were fractionated using a silicic acid column (BOND ELUT LRC-SI; Varian, Palo Alto, CA, USA) and then fatty acid methyl esters were separated from the phospholipids after mild alkaline methanolysis.An internal standard, methyl nonadecanoate (19:0), was added to all samples.Fatty acid methyl esters were identified using the Sherlock Microbial Identification System (MIDI, Newark, DE, USA).The fatty acid 18:2ω6c was used to estimate the biomass of fungi (Frostegård & Baath, 1996); 15:0iso, 15:0antesio, and 17:0iso were used to estimate the Gram-positive bacteria (Olsson & Alström, 2000;Moore-Kucera & Dick, 2008); and 17:0cyclo was used to estimate the Gram-negative bacteria (Wilkinson, 1988).The lipid biomarkers of methanotrophs often overlap with other groups, but in this study the fatty acid 16:1ω7c and 16:1ω5c were used to estimate the biomass of type I methane-oxidizing bacteria and 18:1ω7c was used to estimate the biomass of type II methane-oxidizing bacteria (R. S. Hanson & T. E. Hanson, 1996;Bodelier et al., 2009;Zigah et al., 2015).

Statistical Analysis
The homogeneity of variance and normality of the data collected were determined with Bartlett's test and the Shapiro-Wilk test, respectively.When required, data were log-transformed to meet the assumptions of normality.The main and interaction effects of fertilizers and aquatic earthworms were assessed by generalized linear model (GLM) with Gamma distribution.The effects of the aquatic earthworms on the biomass of methanotrophs were assessed by two-way ANOVA.Statistical analyses were performed using R 3.2.3 for Microsoft Windows (R Development Core Team, 2015) using the package lme4 (Bates et al., 2015) to fit the model.

Soil Environment
The soil Eh value was higher at 1-cm than at 5-cm below soil surface irrespective of treatments throughout the incubation period (Table 1).At 1 cm below soil surface, Eh value was recorded between -35 mV to -73 mV, indicated oxidation reduction potential is much higher than required Eh value to initiate methanogenesis process.In principle, Eh value -150 mV or less leads to a significant CH 4 production.A sharp decrease of Eh values were observed with increasing soil depth.Eh value decreased to -250 mV at 5 cm below soil surface within 7 DAI and same level maintained until the end of the incubation period.The results indicate that Eh value at 5 cm soil depth was favorable for CH 4 formation.Observed soil pH values showed slightly alkaline soil condition over the incubation period irrespective of treatments.Neutral to slightly alkaline pH is beneficial for growth aquatic earthworm (Lou et al., 2013).Table 2 also shows total-C and total-N data of 35 DAI.Total-C ranges from 18.39 g kg -1 in only earthworm treated soil to 19.52 g kg -1 in control treatment (NW NU).However, difference of total-C is not significant among the treatments.Total-N content also showed similar trend.CN ratio was 25:1.It implies that the soil has almost perfect balance of carbon to nitrogen for soil microbial growth.USDA (2011) reported that CN ratio 24:1 is favourable for soil microorganism.

Aquati
The PLFA whereas G amount of  3 and it implies: (i) increasing density of Branchiura sowerbyi has significant positive impact on growth of methanotrophs; (ii) increasing methanotrophs activity leads to significant CH 4 emission reduction; and (iii) as a result Branchiura sowerbyi significantly contributes to mitigate CH 4 in paddy soil environment.

Conclusions
Remarkable increase of atmospheric CH 4 concentration over the centuries has puzzled climate scientists.Flooded rice cultivation has been blamed as one the main sources of anthropogenic CH 4 emission.Anaerobic methanogenesis is the main process of CH 4 production in paddy field soil.It is obvious that wetland rice cultivation will be intensified to feed growing populations, which will also lead increasing contribution to atmospheric CH 4 concentration, our findings showed that bioturbation of aquatic earthworms helps to reduce CH 4 emission to the atmosphere by increasing methanotrophic activities in paddy field soil.Though earlier studies argued that urea application increases CH 4 emission from paddy field, our study showed that in the presence of aquatic earthworms, urea fertilizer reduced CH 4 emission by 90% by stimulating earthworm abundance in soil.Hence, it is important to conserve suitable environment for optimal growth of aquatic earthworms.We also recommend future research should focus on optimization of urea application rate that will be beneficial for earthworm growth and CH 4 oxidation without compromising satisfactory level of crop yield.

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Figure 3. Re

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Figure 5

Table 1 .
The Soil Eh at different depth during incubation period

Table 3 .
Parameter summary results of generalized linear model (GLM)