Dynamics of methanogenesis and methanotrophy in tropical paddy soils as influenced by elevated CO2 and temperature interaction

Soil Biology & Biochemistry 47 (2012) 36e45

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Dynamics of methanogenesis and methanotrophy in tropical paddy soils as influenced by elevated CO2 and temperature interaction Suvendu Das, T.K. Adhya* Laboratory of Soil Microbiology, Division of Crop Production, Central Rice Research Institute, Cuttack 753006, Orissa, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 July 2011 Received in revised form 24 November 2011 Accepted 24 November 2011 Available online 30 December 2011

Response of methanogenesis and methanotrophy to elevated carbon dioxide (CO2) could be affected by changes in soil moisture content and temperature. In soil microcosms contained in glass bottles and incubated under laboratory conditions, we assessed the impact of elevated CO2 and temperature interactions on methanogenesis and methanotrophy in alluvial and laterite paddy soils of tropical origin. Soil samples were incubated at ambient (370 mmol mol
1) and elevated (600 mmol mol
1) CO2 concentrations at 25, 35 and 45  C under non-flooded and flooded conditions for 60 d. Under flooded condition, elevated CO2 significantly increased methane (CH4) production while under non-flooded condition, only marginal increase in CH4 production was observed in both the soils studied and the increase was significantly enhanced by further rise in temperature. Increased methanogenesis as a result of elevated CO2 and temperature interaction was mostly attributed to decreased soil redox potential, increased readily mineralizable carbon, and also noticeable stimulation of methanogenic bacterial population. In contrast to CH4 production, CH4 oxidation was consistently low under elevated CO2 concentration and the decrease was significant with rise in temperature. The low affinity and high affinity CH4 oxidation were faster under non-flooded condition as compared to flooded condition. Admittedly, decreased low and high affinity CH4 oxidation as a result of elevated CO2 and temperature interaction was related to unfavorable lower redox status of soil and the inhibition of CH4-oxidizing bacterial population. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Elevated CO2 Elevated temperature Methanogenesis Methanotrophy Rice paddy

1. Introduction Methane (CH4) and carbon dioxide (CO2) are radiatively important trace gases that are currently increasing in their atmospheric concentration at relative rates of 0.6 and 0.5% year
1 respectively (IPCC, 2007). Increased anthropogenic activity is generally given as the reason for this increase. Methane is 25 times greater relative to the global warming potential of CO2 for a 100 year time horizon (Forster et al., 2007; IPCC, 2007). Flooded rice fields, mostly in the humid tropics, are considered as one of the largest source of atmospheric CH4 with an estimated contribution of approximately 9e19% of the global budget (IPCC, 2007). In freshwater sediments including flooded paddy, about 70% of the biogenic CH4 produced comes from acetate cleavage by acetoclastic methanogens as compared to CO2 reduction by hydrogenotrophic methanogens (Conrad, 2005). In general, acetoclastic methanogenic archeal population predominates in flooded paddy (Conrad,

* Corresponding author. Tel.: þ91 0671 2367757; fax: þ91 0671 2367663. E-mail address: [email protected] (T.K. Adhya). 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.11.020

2007). But in future climate scenario with elevated atmospheric CO2 concentration, hydrogenotrophic methanogenic population can play a more predominant role. Contrary to CH4 production, microbial CH4 oxidation (methanotrophy) is the only known biological sink for atmospheric CH4. The process contributes approximately w80 percent of the produced CH4 being oxidized at the soil surface (Conrad, 2007), where methanotrophic bacteria are able to oxidize CH4 for energy purpose or building up of microbial biomass (Hanson and Hanson, 1996). Two types of kinetics have been described with respect to the concentration of CH4 in soils and sediments (Bender and Conrad, 1992; Nayak et al., 2007a). The first kinetic pattern of CH4 oxidation, known as “low affinity” CH4 oxidation is observed in all CH4-producing soils. This is performed by type I methanotrophs that display Km value in mM range and mainly belong to the class gproteobacteria (Reay, 2003). The second kinetic pattern, known as “high affinity” CH4 oxidation, displays Km value in the nM range and enables oxidation of atmospheric CH4. This process is reported to be mediated by methanotrophs belonging to the class a-proteobacteria, also known as type II methanotrophs (Bull et al., 2000; Dunfield et al., 1999; Lau et al., 2007).

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

The concentration of tropospheric CO2 has been progressively increasing from about 280 ppm at the beginning of the industrial revolution to 379 ppm (IPCC, 2007) at present and future estimates of atmospheric CO2 concentration for the year 2050 range between 450 and 600 ppm (Kattenberg et al., 1996). Coupled with this rise in the concentration of CO2 and other greenhouse gases, an increase in the mean global temperature from 1.4 to 5.8  C by the end of the century (IPCC, 2007) and associated climate change is predicted. There is a strong concern that the climate change will have both direct and indirect effects on terrestrial microbial communities and their functions. The effects of increased CO2 concentrations on microbial communities are often indirect, as they are mediated by effects on plant metabolism, growth and diversity and the associated changes in soil physico-chemical properties such as soil moisture and crop residues (Bardgett and Wardle, 2010). The main direct effect of climate change on soil microorganisms is likely to be caused by changes in temperature and moisture content (Bardgett et al., 2008; Drigo et al., 2009; Garten et al., 2009; Singh et al., 2010). Increasing atmospheric CO2 concentration and rising temperature due to global warming are anticipated to stimulate CH4 emissions from paddy fields. Most studies have shown greater CH4 emissions under high CO2 (Inubushi et al., 2003; Cheng et al., 2006; Lou et al., 2006) and elevated temperature (Inubushi et al., 1984; Ziska et al., 1998; Allen et al., 2003), indicating a positive feedback. Generally speaking, a temperature rise stimulates microbial activity in submerged soils, which may lead to higher rate of CH4 production (Fey and Conrad, 2000). On the other hand several studies from different ecosystems have indicated that CH4 consumption is reduced at elevated levels of CO2 (Phillips et al., 2001a,b; McLain and Ahmann, 2008; Dubbs and Whalen, 2010), but the causative mechanisms are not well known. It is possible that CO2 enrichment affects the size or activity of the CH4-oxidizing microbial community or causes a higher soil C concentration and competition for O2 and therefore suppression of the CH4-oxidizing microbial community (Phillips et al., 2001b). Increased soil moisture under elevated CO2 reduces the rate of diffusion and therefore decreases CH4 oxidation in the soil (Ambus and Robertson, 1999; Phillips et al., 2001a). However, if the rising temperature due to the global climate change makes the soil drier, CH4 oxidation may be enhanced (Dijkstra et al., 2010). Most experimental designs till date have studied the impact of single climate variable (e.g. increased temperature) on CH4 cycling. However, for a more accurate explanation it is essential to investigate microbial responses in multi-factorial experimental designs in which several interacting climatic variables could be studied (Singh et al., 2010). We, therefore, took advantage of a multi-factor experiment in two physico-chemically different tropical paddy soils to better understand the effect and interaction of increased CO2 and warming on methanogenesis and methanotrophy. The objective of this research was to study the impact of elevated CO2 and temperature interaction on (i) methanogenesis, (ii) methanotrophy and (iii) associated soil biochemical parameters and microorganisms involved.

37

2. Materials and methods 2.1. Soils The experiment was carried out with two tropical paddy soils. Soil samples (surface, 0e15 cm) were collected from the rice fields of the experimental farm of the Central Rice Research Institute, Cuttack (20 250 N, 85 550 E), Cuttack, Orissa and farmer’s field at Sukinda (20 580 N, 85 540 E), Orissa, in the month of May (fallow period between riceerice cropping system), air dried in shade with appropriate care for avoiding contamination from other soil samples and airborne dust, grounded, sieved (>2 mm) and stored in polyethylene bags at room temperature under dry condition. Physico-chemical parameters of the soils were determined according to Sparks et al. (1996). In brief, the soil pH was measured at soil:water ratio of 1:1.25 using a portable pH meter (Philips model PW 9424) with a combined calomel glass electrode assembly and expressed as the negative logarithm to base 10 of the H ion activity. Electrical conductivity (EC) measured at soil:water ratio of 1:1.25 using a conductivity bridge (Elico India, Model CM180). Total Fe content was estimated by extracting the soil with sodium dithionite and then reacting the resulting extractant with KCNS. Moisture holding capacity (MHC) of the soils was determined in 10 g portions of soil by gravimetric analysis and expressed in percent (Fierer and Schimel, 2002). Cation exchange capacity (CEC) was determined by summation of exchangeable Na, K, Ca, Mg and H. The organic carbon content of the soil samples were determined in a TOC analyzer (Micro N/C model HT 1300, Analytic Jena, Germany) while Total N was analyzed by a semi-automated Kjeldahl method (Kjeltech model 2100, Foss Tecator, Sweden) (Nayak et al., 2007a). Clay, silt and sand contents were analyzed by employing the Bouyoucos hydrometer method. The data are reported in Table 1. 2.2. Experimental set-up Experiments were set-up for a period of 60 d under two moisture regimes (flooded and non-flooded), by placing 20 g soil in 130 ml serum bottles pre-sterilized in an autoclave at 120 kPa for 1 h, and closed with neoprene septa. For the flooded experiment, 20 g soils were flooded with 25 ml sterile distilled water (soil:water: 1:1.25) to provide a thin layer (1 cm) of standing water. For the non-flooded experiment, soils were moistened with sterile distilled water to 60% MHC. Elevated CO2 concentrations in the headspace were set at 600  20 mmol mol
1 by replacing 30 ml of air in the headspace with the equivalent amount of 0.2% CO2 in air. Soil samples in serum bottles were incubated at 25, 35 and 45  C in separate BOD incubators for the duration of the experiment. Changes in CO2 concentration in the serum bottles, if any, were monitored at 2 d intervals by sampling 5 ml of headspace gas and quantifying the CO2 content by absorption in 0.1 N NaOH. The CO2 concentration was kept constant by injecting the required quantity of CO2 in the headspace of the serum bottles. Changes in CO2 concentration were never higher than 2% (Das et al., 2011a). Appropriate control flasks with soil under either flooded or non-

Table 1 Physico-chemical properties of soils. Location

Cuttack Sukinda

Soil type

Alluvial Laterite

pH

6.16 5.90

EC (ds m
1)

CEC (meq g
1 soil)

Organic carbon (%)

Total nitrogen (%)

Total Fe content (mg g
1)

Moisture holding capacity (%)

Soil separates Clay (%)

Silt (%)

Sand (%)

0.50 1.10

15.0 6.0

0.86 0.76

0.09 0.04

8.62 11.92

47.1 36.8

25.9 14.6

21.6 10.6

52.5 74.8

38

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

flooded conditions were maintained with ambient air (CO2 concentration 380 mmol mol
1) in the headspace. In the present study using soils from locations where the highest ambient temperature shoots to w45  C during the peak summer months (AprileJune), we introduced the treatment as the extreme range.

associates Inc., USA) as primary standard and 2.14 ml CH4 ml
1 in air as secondary standard to provide a standard curve linear over the concentration range used. Under these conditions, the retention time of CH4 was 0.53 min and the minimum detectable limit was 0.5 ml ml
1 (Nayak et al., 2007a; Das et al., 2011b).

2.3. CH4 production

2.6. Soil physico-chemical analysis

CH4 production was quantified by withdrawing 1 ml air sample from the headspace of the serum bottles at 5 d interval (Adhya et al., 2000) and quantified by gas chromatography as described below. The amount of CH4 produced under laboratory incubation was expressed as nanogram (ng) of CH4 g
1 dry soil, based on the calculation:

Redox potential and pH of laboratory-incubated soil samples under flooded and non-flooded conditions were determined in duplicates. For determining the redox potential, separate duplicate sets of 20 g soil incubated in 130 ml serum bottles under varying concentrations of CO2 and temperature were used. The redox potential was measured with a Barnant-20 digital ORP meter (Model 559-3800, Barnant Company, Barrington, Illinois, USA) fitted with a platinum electrode. The electrode was placed 1e2 cm deep into the reduced soil layer and the redox potential was read in the potentiometer till it gave a stable reading for 10 s. The measurements were made in mV. Before recording the actual soil redox measurements, the redox meter was standardized with a solution of 0.0033 M K3FeCN6 and 0.0033 M K4FeCN6 in 0.1 M KCl which has an Eh of þ0.222 V at 25  C. After measuring the redox potential, pH of the soil was measured by inserting a combined pH electrode (UNICAM combination electrode, Pye-Unicam, UK). For non-flooded soil, pH was measured after adding distilled H2O in 1:25 ratio. The pH electrode was connected to a potentiometer (Philips PW 9424, Philips, UK) and the pH measured.

ng of CH4 g
1 soil ¼

CHS  VHS  r Ws

where CHS ¼ concentration of CH4 in the headspace (calibrated against standard CH4), VHS ¼ volume of headspace of the bottle, r ¼ density of CH4 at a given temperature [r ¼ (MW  TST)/ (MV  (TST þ T)] where MW ¼ 16.123  10
3 mg, MV ¼ 22.41  10
3 m2, T ¼  C, TST ¼ 273.2 K], and Ws ¼ dry weight of the soil. 2.4. CH4 oxidation High affinity and low affinity CH4 oxidations were measured in flooded and non-flooded soils samples under different concentrations of CO2 and temperatures at the end of the incubation. High affinity CH4 oxidation was initiated by sealing the serum bottles with neoprene septa and injecting the headspace with 1 ml of pure CH4 to provide approximately 2 mL L
1 CH4 (Topp and Hanson, 1991). Low affinity CH4 oxidation was initiated by sealing serum bottles and injecting the headspace with 10 ml of CH4 to provide approximately 40 mL L
1 CH4 (King et al., 1990; Jones and Nedwell, 1993). Headspace gas samples (5 ml) of the serum bottles were analyzed for CH4 until 10 d, initially at an interval of 24 h up to 5 d and at an interval of 2 d up to 10 d. After each sampling, the headspace was replaced with an equivalent amount of high-purity Ar to maintain the pressure equilibrium (Nayak et al., 2007a). The amount of CH4 remaining in the headspace was plotted on a log scale against the time of incubation (Alexander, 1994). The decomposition of CH4 followed a first-order reaction as the plots yielded straight line based on the equation:

C ¼ C0 e
kt where C is the concentration of CH4 remaining in the vial after time t, C0 is the initial concentration of CH4, and k is the first-order kinetic constant. The half-life (t1/2) values obtained from these plots indicated the oxidation rate of CH4 in days. 2.5. Quantification of CH4 CH4 content in the headspace of the incubated serum bottles was quantified by gas chromatography in a Varian 3600 gas chromatograph equipped with FID and Porapak N column (2 m length, 1/8 in OD, 80/100 mesh, stainless steel column). The column, injector and detector were maintained at 70, 80 and 150  C respectively. The desired amount of gas samples was withdrawn from the headspace of serum bottles by using a precision gas sampling syringe. High-purity nitrogen gas maintained at 30 ml min
1 was used as carrier gas. The gas chromatograph was calibrated before and after each set of measurements using 5.38, 9.03 and 10.8 ml CH4 ml
1 in N2 (ScottyÒ II analyzed gases, Altech

2.7. Microbiological analysis Methanogen and methanotroph populations were enumerated at the end of the incubation. Methanogenic bacterial populations were enumerated following anaerobic culture tube technique (Kasper and Tiedje, 1982; Mohanty et al., 2009). The medium used for enumeration of methanogens consisted of the following components (mM): KH2PO4 (5.00), K2HPO4 (4.99), MgSO4.7H2O (0.16), cysteine HCl. H2O (3.17), NH4Cl (18.70), NaHCO3 (1.19) and Na2S.9H2O (1.04); yeast extract (2 g), 1 ml of resazurin solution (Kasper and Tiedje, 1982) and 20 ml l
1 each of vitamin and mineral solutions. The anaerobic MPN tubes, prepared under N2 gas, were either pressurized with 2 ml of CO2/H2 (2:1, by vol.) (for hydrogenotrophic methanogens) or supplemented with sodium acetate to give a final concentration of 10 mmol (for acetoclastic methanogens) and incubated at 28 þ 2  C for 30 d. Detection of more than 2 ml ml
1 of CH4 in the headspace of culture tubes was considered as evidence for the presence of methanogens and the population was counted by most probable number (MPN) method. Methane oxidizers with soluble methane monooxygenase (sMMO) activity were enumerated as described by Graham et al. (1992). The soil samples were prepared by serial dilution in copper-free 0.1% inorganic pyrophosphate buffer and then plated directly onto copperfree Noble agar (Difco laboratories) nitrate salts medium plates. Triplicate plates for each dilution were incubated in vacuum desiccator under the atmosphere of CH4 (5%)-air mixture by replenishing the headspace atmosphere with CH4 on every 4 d, for 30 d in an incubator at 35  C. The colonies that developed a colored complex with naphthalene and O-dianisidine (tetrazotized) were counted positive for CH4 oxidizers with sMMO (Mohanty et al., 2009). 2.8. Statistical analysis Data were subjected to statistical analysis (Gomez and Gomez, 1984) by a statistical package (IRRISTAT version 3.1; International Rice Research Institute, Los Banos, Philippines). The mean

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

difference comparison between the treatments was analyzed by analysis of variance (ANOVA) and subsequently by Duncan’s multiple range test (DMRT) at p < 0.05.

39

45  C the effect increased up to 20 d and decreased thereafter. However, under non-flooded condition, the CO2 enrichment effect on CH4 production increased up to 45 d and decreased thereafter at 25  C and 35  C, while at 45  C, the treatment effect increased up to 20 d and decreased afterward (Fig. 2).

3. Results 3.1. CH4 production

3.2. CH4 oxidation

Elevated CO2 significantly (p < 0.001) increased CH4 production and was further enhanced (p < 0.001) by increase in temperature. CH4 production in control and CO2-enriched soil followed the same pattern at 25, 35 and 45  C under flooded and non-flooded conditions in both alluvial as well as laterite soils, but the magnitude of CH4 production varied. CH4 production increased up to 45 d of incubation and decreased thereafter (Fig. 1). In both the alluvial and laterite soils under the two moisture regimes, cumulative CH4 production was higher in CO2-enriched soil over that of the corresponding control incubated at 25, 35 and 45  C respectively (Table 2). CO2 concentration significantly interacted with both temperature and water regimes but CO2 concentration exerted higher interaction with flooding than temperature (Table 3). Polynomial function provided the best fit to elevated CO2 response for CH4 production under flooded and non-flooded soils at 25 , 35 and 45  C (Fig. 2). Interestingly, during the 60 d incubation under flooded condition, the effect of CO2 enrichment on CH4 production gradually increased up to 60 d at both 25  C and 35  C, while at

CO2 enrichment significantly (p < 0.001) decreased both low and high affinity oxidation. Rise in temperature also inhibited (p < 0.001) both low and high affinity CH4 oxidation. However, CO2 enrichment did not significantly interact with temperature both in case of low affinity and high affinity CH4 oxidation (Table 3). Furthermore, high affinity CH4 oxidation was not detected in flooded soil, as flooded soils are net CH4 producers. k value for low affinity CH4 oxidation in CO2-enriched soil was significantly lower than the control soil incubated at 25, 35 and 45  C both under flooded as well as non-flooded conditions. Similarly, k value for high affinity CH4 oxidation in CO2-enriched soil was also significantly lower than the control soil incubated at 25  C under nonflooded condition (Table 4). In alluvial soil, under flooded condition, low affinity CH4 oxidation was 26.10, 28.54 and 33.64% lower while under non-flooded condition, it was 5.67, 9.50 and 16.21% lower under CO2 enrichment than in control at 25, 35 and 45  C respectively. Similarly in laterite soil under flooded condition, low affinity CH4 oxidation was 14.94, 19.79 and 26.32% lower, whereas

A 500

Alluvial 25º C

150

200

100

100

CH4 production (ng g-1 soil )

50 0 5 10 15 20 25 30 35 40 45 50 55 60

35º C

35º C

300

150

200

100

100

50 0 5 10 15 20 25 30 35 40 45 50 55 60

45º C

0

0 5 10 15 20 25 30 35 40 45 50 55 60

45º C

300 250

400

200

300

150

200

100

100 0

0 5 10 15 20 25 30 35 40 45 50 55 60

200

300

500

0

250

400

0

25º C

300 200

300

500

Laterite

250

400

0

B

50 0 5 10 15 20 25 30 35 40 45 50 55 60

0

0 5 10 15 20 25 30 35 40 45 50 55 60

Days of incubation Fig. 1. CH4 production of A) alluvial and B) laterite soils at 25, 35 and 45  C under flooded and non-flooded conditions as influenced by CO2 enrichment. Mean of three replicate values plotted, bars/half-bars indicate the standard deviation (---: elevated CO2 and flooded, -A-: ambient CO2 and flooded, --: elevated CO2 and non-flooded, –: ambient CO2 and non-flooded).

40

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

Table 2 Effect of elevated CO2 and temperature on cumulative CH4 production under flooded and non-flooded conditions. Soil type

CO2 enrichment

Cumulative CH4 production (ng g
1)a 25  C

Alluvial Laterite

Control Elevated Control Elevated

35  C

Non-flooded

Flooded

762.81 832.44 536.49 586.09

1994.80 2236.76 1592.63 1848.02

a b (9.1) a b (9.2)

a b (16.2) a b (16.0)

45  C

Non-flooded

Flooded

1033.22 a 1144.59 b (10.8) 692.57 a 765.15 b (10.5)

2548.00 3047.73 1889.24 2239.66

a b (19.6) a b (18.5)

Non-flooded

Flooded

1231.77 a 1396.94 b (13.4) 973.76 a 1100.79 b (13.0)

3469.80 4214.40 2192.88 2646.39

a b (21.5) a b (20.7)

a Mean of three replicate observations. Means of cumulative CH4 production values in a column under each soil type with same letter do not differ significantly (p < 0.05) by Duncan’s Multiple Range Test (DMRT). Values in parenthesis indicate per cent increase in cumulative CH4 production under elevated CO2 (600 mmol mol
1) over the corresponding control (380 mmol mol
1).

under non-flooded condition it was 5.32, 7.19 and 13.38% lower under CO2 enrichment than in control at 25, 35 and 45  C respectively. Percentage decrease in high affinity CH4 oxidation as a result of CO2 enrichment effect was 6.14, 5.81 and 8.89 in alluvial soil and 6.67, 9.63 and 7.44 in laterite soil under non-flooded condition at 25, 35 and 45  C respectively. 3.3. Soil properties Carbon dioxide enrichment significantly (p < 0.001) decreased the redox potential (Eh) of soil. Rise in temperature also significantly (p < 0.001) decreased Eh of soils. Elevated CO2 significantly interacted with temperature and water regime for Eh of the soil and the interaction of elevated CO2 with moisture regime was more pronounced than the CO2 and temperature interaction (Table 3). Polynomial function provided the best fit to elevated CO2 response for Eh under flooded and non-flooded soils at 25, 35 and 45  C (Fig. 3). Among the three temperatures tested, the treatment effects was more both under flooded and non-flooded condition at 45  C. In the 60 d incubation study, CO2 enrichment effect increased up to 50 d, than decreased gradually depending on moisture regime, temperature of incubation and soil type (Fig. 3). Like Eh, elevated CO2 significantly (p < 0.001) decreased the pH of soil and was further decreased (p < 0.001) by an increase in temperature. The interaction of elevated CO2 with temperature (p < 0.01) and flooding (p < 0.001) was also significant (Table 3). In the present study, both in flooded and non-flooded conditions, pH of the soil stabilized near neutrality during initial stages of incubation but reverted back to acidic situations upon further incubation. Polynomial function providing the best fit to elevated CO2 response for pH under flooded and non-flooded soils at three temperatures, revealed that CO2 enrichment effect was more pronounced in flooded soil than the non-flooded soil and among the three temperatures, the treatment effects was more at 45  C both under flooded and non-flooded condition (Fig. 4). Interestingly, both in alluvial and laterite soils, under flooded and nonflooded condition at 25, 35 and 45  C, the CO2 enrichment effect

on pH increased up to 40 d of incubation and decreased thereafter (Fig. 4). 3.4. Microbial population Elevated CO2 significantly (p < 0.001) enhanced the population of acetoclastic methanogen population and was further significantly (p < 0.001) enhanced by rise in temperature (Table 3). Significant increase in acetoclastic methanogens population as a result of CO2 enrichment effect was observed at 35 and 45  C under flooded condition only (Table 5). In the alluvial soil, increase in the population of acetoclastic methanogens as a result of CO2 enrichment over control, was 30.7, 39.6 and 40.9% at 25, 35 and 45  C respectively under flooded condition, while under nonflooded condition, the percentage increase was 17.7, 20.3 and 23.7 at 25, 35 and 45  C respectively. In laterite soil, under flooded condition, the percentage increase in acetoclastic methanogen population due to the elevated CO2 effect over control was 26.6, 32.6 and 38.9 at 25, 35 and 45  C respectively, whereas under nonflooded condition the percentage increase was 15.1, 17.7 and 23.4 at 25, 35 and 45  C respectively. Results of statistical analysis showed that elevated CO2 significantly interacted with flooding (p < 0.001), while the interaction of elevated CO2 and temperature was not significant (Table 3). Though the population of hydrogenotrophic methanogens was lower as compared to acetoclastic methanogens in all the treatments, the stimulation of the former as a result of CO2 enrichment was much more favored than the later (Table 3, Table 5). Significant increase in hydrogenotrophic methanogen population as a result of CO2 enrichment was observed at 25, 35 and 45  C under flooded condition only (Table 5), indicating flooding the soil responded more positively to the CO2 enrichment and favored significant stimulation of the methanogenic population. In alluvial soil, under flooded condition, the percentage increase in hydrogenotrophic methanogen population due to the elevated CO2 effect over control was 38.1, 44.1 and 46.2 at 25, 35 and 45  C respectively, whereas under non-flooded condition the percentage increase was 22.2,

Table 3 F values of CH4 production, CH4 oxidation, soil properties and microbial parameters as influenced by the analysis of variance (ANOVA factors were CO2 concentration, temperature, moisture regime and soil type). Sources of Variation

CH4 production

CH4 oxidation (low affinity)

CH4 oxidation (high affinity)

Eh

pH

M1

M2

M3

C T W CT CW TW CTW

6063** 21,140** 207,699** 286.7** 2400** 3841** 92.43**

440** 1751** 8948** 1.62 ns 18.2** 661** 0.70 ns

8.91** 84.7** nd 0.01 ns nd nd nd

324** 743** 24,120** 4.49* 15.2** 73.2** 0.04 ns

1475** 53.5** 127** 3.88* 64.9** 0.25 ns 0.16 ns

18.5** 33.7** 645** 1.42 ns 16.3** 28.9** 1.27 ns

39.4** 56.6** 1064** 2.47 ns 33.3** 47.1** 2.17 ns

152** 34.0** 105** 2.28 ns 14.6** 0.69 ns 0.14 ns

C: CO2 concentration; T: Temperature; W: Water regime; M1: Methanogens (acetotrophs), M2: Methanogens (hydrogenotrophs), M3: Methanotrophs. *Significant at p < 0.05; **Significant at p < 0.001; ns: not significant; nd: not detected.

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

A Treatment effect (ng CH4 g-1 soil )

B

25º C

35º C

45º C

80

R ² = 0.988

80

60

R ² = 0.802

60

60

40

40

40

20

20

0

0

0

10 20 30 40 50 60

R ² = 0.945

R ² = 0.923

0

35º C

R ² = 0.992

80

60

R ² = 0.808

60

80

R ² = 0.987 R ² = 0.839

0

40

20

20

20

0

0

45º C R ² = 0.803 R ² = 0.624

60

40

10 20 30 40 50 60

R ² = 0.638 0 10 20 30 40 50 60

80

40

0

R ² = 0.763

20

10 20 30 40 50 60

25º C

80

0

41

10 20 30 40 50 60

0

0

10 20 30 40 50 60

Days of incubation Fig. 2. Effect of CO2 enrichment on CH4 production in A) alluvial and B) laterite soil at 25, 35 and 45  C under flooded and non-flooded conditions. The treatment effect on each incubation day is calculated as the difference between the mean rate of CH4 production in CO2-enriched soil minus mean rate of CH4 production in ambient soil (-:-: flooded; ---: non-flooded). In each figure upper R2 value represents flooded soil and lower R2 value represents non-flooded soil.

24.0 and 26.0 at 25, 35 and 45  C respectively. In laterite soil, under flooded condition, increase in hydrogenotrophic methanogen population as a result of CO2 enrichment effect over control was 33.3, 38.7 and 42.7% at 25, 35 and 45  C respectively, whereas under non-flooded condition, the percentage increase was 19.2, 21.2 and 25.7 at 25, 35 and 45  C respectively. Statistical analysis showed that elevated CO2 significantly (p < 0.001) stimulated hydrogenotrophic methanogen population and was further enhanced (p < 0.001) by rise in temperature. Elevated CO2 significantly interacted with flooding (p < 0.001), while the interaction of elevated CO2 and temperature was not significant (Table 3). Unlike methanogenic bacteria, methanotrophic bacterial population decreased significantly (p < 0.001) at elevated CO2 concentration and was further significantly (p < 0.001) influenced by elevated temperature (Table 3). Compared to flooded condition,

methanotrophic bacterial population proliferated and reached maximum at 35  C under non-flooded condition (Table 5). In alluvial soil under flooded condition, the percentage decrease in methanotrophic bacterial population as a result of CO2 enrichment was 31.7, 33.1 and 34.2 at 25, 35 and 45  C respectively, whereas under non-flooded condition the percentage decrease was 37.7, 40.4 and 42.2 at 25, 35 and 45  C respectively and was significant. In laterite soil, under flooded condition, decrease in methanotrophic bacterial population as a result of CO2 enrichment was 22.8, 28.5 and 36.1% at 25, 35 and 45  C respectively, whereas under nonflooded condition, the percentage decrease was 35.9, 36.1 and 37.7 at 25, 35 and 45  C respectively and were significant. Elevated CO2 significantly interacted with moisture regimes (p < 0.001), while the interaction of elevated CO2 and temperature was not significant (Table 3).

Table 4 Effect of elevated CO2 and temperature on low and high affinity oxidation of CH4 under flooded and non-flooded condition. Soil type & temperature

Moisture regimes

CH4 oxidationa Low affinity

High affinity t1/2 (d)

k value

Alluvial 25  C 

35 C 45  C Laterite 25  C 

35 C 45  C

k value

t1/2 (d)

Ambient CO2

Elevated CO2

Ambient CO2

Elevated CO2

Ambient CO2

Elevated CO2

Ambient CO2

Elevated CO2

F NF F NF F NF

0.174 0.423 0.151 0.337 0.144 0.193

b b b b b b

0.128 0.399 0.107 0.305 0.095 0.162

a a a a a a

4.0 a 1.64 a 4.60 a 2.06 a 4.89 a 3.60 a

5.40 1.74 6.44 2.27 7.28 4.29

b a b a b b

ND 0.076 b ND 0.058 a ND 0.045 a

ND 0.071 a ND 0.054 a ND 0.041 a

ND 9.17 a ND 12.38 a ND 15.55 a

ND 9.77 a ND 12.91 a ND 16.96 b

F NF F NF F NF

0.143 0.219 0.096 0.199 0.095 0.152

b b b b b b

0.121 0.208 0.077 0.185 0.070 0.132

a a a a a a

4.84 3.18 7.22 3.49 7.29 4.57

5.72 3.34 9.00 3.75 9.00 5.29

b a b a b b

ND 0.050 a ND 0.045 a ND 0.041 a

ND 0.047 a ND 0.041 a ND 0.037 a

ND 13.79 a ND 15.56 a ND 17.14 a

ND 14.86 a ND 17.09 b ND 18.74 b

a a a a a a

F: Flooded, NF: Non-flooded, ND: Not detected. a Mean of three replicate observations. Means of k value, t1/2 value in low affinity and high affinity CH4 oxidation, in a row with same letter do not differ significantly (p < 0.05) by Duncan’s Multiple Range Test (DMRT).

42

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

A

25º C

40

B

Treatment effect (Soil Eh in mV )

30

R ² = 0.967

R ² = 0.961

30 20

20 10 0

35º C

40

R ² = 0.780 0

10 20

30 40

50 60

R ² = 0.709 0

10

20 30 40

40

R ² = 0.928

30 20

R ² = 0.947

30

0

10 20

30 40

50 60

0

10

20 30 40

0

50 60

45º C 40

R ² = 0.784

20

R ² = 0.713

10

R ² = 0.842

R ² = 0.544

0

30

20

10 0

50 60

10

35º C

25º C 40

R ² = 0.735

30 20

10 0

45º C

40

0

10 20

30 40

50 60

R ² = 0.548

10 0

0

10 20

30 40

50 60

Days of incubation Fig. 3. Effect of CO2 enrichment on Eh of A) alluvial and B) laterite soil at 25, 35 and 45  C under flooded and non-flooded conditions. The treatment effect on each incubation day is calculated as the difference between the mean Eh in CO2-enriched soil minus mean Eh in ambient soil (-:-: flooded; ---: non-flooded). In each figure upper R2 value represents flooded soil and lower R2 value represents non-flooded soil.

4. Discussion Among the many factors that may affect methanogenesis and methanotrophy in paddy soils, temperature and moisture have a special influence (Parashar et al., 1993; Bharati et al., 2001; Rath et al., 2002). In the present laboratory incubation study, it was observed that interaction of elevated CO2 with temperature and flooding significantly enhanced CH4 production. However, the interaction of elevated CO2 with flooding exerted greater influence than the interaction of elevated CO2 and temperature on CH4 production (Table 3). The increase in temperature accelerates the decomposition of organic matter (Kirschbaum, 1995; von Lützow

A

25º C

1.00 0.80

R ² = 0.829

B

Treatment effect (Soil pH)

0.60

1.00 0.80 0.60

0.40

0.40

0.20

0.20

R ² = 0.783

0.00 -0.20 0

0.00 10 20 30 40 50 60 -0.20 0

35º C R ² = 0.863

25º C

0.40

R ² = 0.809

R ² = 0.787

0.20

R ² = 0.811

0.20

R ² = 0.768

0.00 10 20 30 40 50 60

45º C

0.40

R ² = 0.889

R ² = 0.676

R ² = 0.662 0.00

10 20 30 40 50 60

R ² = 0.796

0.60

0.20

R ² = 0.676 0

45º C

-0.40

0.20

0.00

0.80

10 20 30 40 50 60 -0.20 0

35º C

0.40

1.00

0.40

-0.40

-0.40

-0.20

and Kogel, 2009; Karhu et al., 2010) and CO2 enrichment further stimulates organic matter decomposition (Cheng and Johnson, 1998; Carney et al., 2007; Huttunen et al., 2009). The interaction of CO2 enrichment with temperature may exert greater stimulation of organic matter decomposition, which in turn provides substrates for methanogenic bacteria. Besides temperature, floodwater regime is the other most important determinant for CH4 production and emission from rice fields (Yagi et al., 1996; Adhya et al., 2000; Bharati et al., 2001). Flooding the soil hastens creating anaerobic environment resulting from the depletion of the porespace oxygen by heterotrophic microbial respiration and subsequent limitation in the supply of O2 from the atmosphere (Conrad, 2007). The

0.00 0

10 20 30 40 50 60

-0.20

0

10 20 30 40 50 60

-0.20

Days of incubation Fig. 4. Effect of CO2 enrichment on pH of A) alluvial and B) laterite soil at 25, 35 and 45  C under flooded and non-flooded conditions. The treatment effect on each incubation day is calculated as the difference between the mean pH in ambient soil minus mean pH in CO2-enriched soil (-:-: flooded; ---: non-flooded). In each figure upper R2 value represents flooded soil and lower R2 value represents non-flooded soil.

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

43

Table 5 Effect of elevated CO2 and temperature on methanogens and methanotrophs bacterial population under flooded and non-flooded condition. Soil types

Moisture regimes

Microbial population ( 104 g
1 soil)a Methanogens (acetoclastic) Ambient CO2

Alluvial 25  C 

35 C 45  C Laterite 25  C 

35 C 45  C

Elevated CO2

Methanogens (hydrogenotrophs)

Methanotrophs

Ambient CO2

Ambient CO2

Elevated CO2

Elevated CO2

F NF F NF F NF

1.73 0.12 2.33 0.13 3.13 0.20

     

0.06 0.01 0.12 0.02 0.64 0.02

a a a a a a

2.27 0.13 3.27 0.16 4.40 0.24

     

0.12 0.01 0.40 0.02 0.95 0.03

a a b a b a

1.13 0.08 1.60 0.12 2.17 0.14

     

0.06 0.01 0.17 0.01 0.40 0.03

a a a a a a

1.57 0.11 2.30 0.15 3.17 0.18

     

0.12 0.01 0.17 0.02 0.58 0.04

b a b a b a

8.3 12.3 11.3 17.3 10.7 15.0

     

1.15 1.15 1.53 1.53 0.53 2.00

b b b b b b

5.7 7.7 7.0 10.3 7.0 8.7

     

1.15 1.15 2.08 1.15 0.12 1.53

a a a a a a

F NF F NF F NF

1.23 0.09 1.86 0.12 2.77 0.13

     

0.12 0.02 0.29 0.01 0.64 0.02

a a a a a a

1.57 0.10 2.47 0.13 3.90 0.16

     

0.23 0.01 0.31 0.01 1.35 0.02

a a b a b a

0.95 0.08 1.23 0.10 1.93 0.13

     

0.00 0.00 0.12 0.01 0.40 0.02

a a a a a a

1.27 0.09 1.70 0.13 2.77 0.16

     

0.06 0.00 0.00 0.02 0.64 0.02

b a b a b a

5.7 9.3 9.3 13.7 7.7 12.3

     

1.15 0.58 1.53 3.06 1.15 1.15

a b b b a b

4.3 6.0 6.7 8.7 5.3 7.7

     

0.58 1.00 1.15 1.53 0.58 0.58

a a a a a a

F: Flooded, NF: Non-flooded. a Mean of three replicate observations  Standard deviation. Means in a row in different microbial population with same letter do not differ significantly (p < 0.05) by Duncan’s Multiple Range Test (DMRT).

interaction of elevated CO2 with flooding further stimulated anoxia favoring methanogenesis. Methanogenesis has been linked to low redox potential and a near neutral pH of inundated soil (Masscheleyn et al., 1993). In this study, elevated CO2 significantly decreased soil redox potential and was further decreased significantly by rise in temperature. Correlation analysis between CH4 production and soil redox potential showed highly significant negative relationship (r ¼
0.917, p < 0.001). At the same time correlation analysis between CH4 production and soil pH showed significant positive relationship (r ¼ 0.730, p < 0.05) indicating redox potential and soil pH, influenced by CO2 enrichment, were important parameters governing methanogenesis. Increase in methanogenic bacterial population as a result of CO2 enrichment effect was the prime reason behind the enhanced CH4 production at elevated CO2 concentration. The traditional method for enumerating methanogens and methanotrophs in environmental samples may be limited by cultivation bias but is still widely used (McDonald et al., 2008). In the present study, it was observed that elevated CO2 significantly (p < 0.001) increased both, acetoclastic and hydrogenotrophic methanogen population and was further significantly (p < 0.001) enhanced by a rise in temperature. Compared to non-flooded soil, proliferation of methanogen population due to CO2 enrichment effect was higher in flooded soil. This may be attributed to lower redox potential of flooded soil than the non-flooded soil, which would have favored the proliferation of methanogens. In flooded rice soils, hydrogenotrophic methanogens are noticed during the initial phase of flooding which is usually taken over by acetoclastic methanogens at later stages (Ramakrishnan et al., 1998; Conrad, 2007). However, in the present study the percentage increase of hydrogenotrophic methanogen population was more than that of acetate utilizing methanogens at elevated CO2 concentration, suggesting CO2 may be a limiting factor for methanogenesis by this group of bacteria. The increase in methanogen population is most likely caused by the increase in substrate availability for methanogens under the condition of elevated CO2. In an earlier study we found that elevated CO2 significantly enhanced readily mineralizable carbon (Das et al., 2011a). Paterson et al. (2008) in a 13C labeling experiment showed that atmospheric CO2 enrichment and nutrient additions to planted soil increase mineralization of soil organic matter. Increased readily mineralizable carbon (RMC) content, an indicator of readily available C for microbial metabolism (Yagi and Minami,

1990; Nayak et al., 2007b), may trigger the proliferation of methanogens and methanogenesis at elevated CO2 concentration. Rates of CH4 consumption tend to increase as soil moisture declines and temperature rises (van den Pol-van Dasselaar et al., 1998; Phillips et al., 2001b). In the present study, flooding significantly (p < 0.001) decreased CH4 consumption and the decrease was significant (p < 0.001) with rise in temperature. Knowles (1993) suggested that CH4 oxidizers are generally mesophilic in nature, with an optimum of 20e30  C. The optimum temperature of CH4 oxidation is lower than the optimum temperature for CH4 production (Conrad et al., 1987). In the experiment elevated temperature might hinder the growth of methanotrophs, causing decreased CH4 consumption. CH4 oxidation is mostly anaerobic process (Conrad, 1995). Soil anoxia caused by flooding greatly reduced CH4 oxidation. Flooded rice soil being a net producer of CH4, low affinity CH4 oxidation is predominant over high affinity CH4 oxidation (King, 1994; Nayak et al., 2007a). In this experiment low affinity oxidation dominated over high affinity oxidation and was significantly (p < 0.001) decreased by CO2 enrichment. Decreased methanotrophic bacterial population to elevated CO2 and temperature, may be the reason of decreased methanotrophy in this CO2 enrichment study. Methanotrophic bacteria with sMMO are mostly autotrophic bacteria utilizing C-1 compounds for their metabolic activity and autotrophic bacterial population are mostly affected by environmental influences. However, reduced methanotrophic activity under elevated CO2 concentration was considered to represent acclimatization rather than equilibration (Phillips et al., 2001a). Down-regulation or a reduced metabolic response to treatment has been reported for some biological processes in other CO2-enriched environment (Tissue et al., 1997). CO2 enrichment has altered soil microbial community by increasing soil carbon (Zak et al., 1993; Luo et al., 2006) and competition for O2, which may have resulted in the repression of the CH4-oxidizing population or activity levels (Phillips et al., 2001a). Greater soil respiration under elevated CO2 (Allen et al., 2000; Jackson et al., 2009; Deng et al., 2010) may have lower O2 availability, thereby inhibiting the oxidation of CH4 by methanotrophs. The findings of this study can be summarized in the following way: (1) Elevated CO2 significantly increased CH4 production and the increase was significant with the rise in temperature. Thus, the interaction of elevated CO2 and temperature had significant impact on CH4 production. (2) Elevated CO2 significantly decreased both

44

S. Das, T.K. Adhya / Soil Biology & Biochemistry 47 (2012) 36e45

low and high affinity CH4 oxidation of soil and the decrease was significant over rise in temperature, albeit the interaction of elevated CO2 and temperature on low and high affinity CH4 oxidation being not significant. (3) CO2 enrichment significantly decreased redox potential and pH of soil and was further significantly decreased by rise in temperature. The interaction of elevated CO2 and temperature was significant. (4) Methanogen population was significantly enhanced by CO2 enrichment and was further significantly enhanced by rise in temperature. (5) Methanotroph population significantly decreased by CO2 enrichment effect and the decrease was significant over rise in temperature. (6) The flooded soil responded more positively to the CO2 enrichment than the non-flooded soil. Higher rates of CH4 production and lower rates of CH4 oxidation for soils exposed to elevated CO2 and temperature as compared to ambient atmospheric CO2 (control), as observed in the present study, is evidence of altered ecosystem function as a result of interactive effect of elevated CO2 and temperature. Possible longterm consequences of a rising atmospheric CO2 and temperature may be a reduction in the strength of the CH4 soil sink and a positive feedback to the greenhouse effect. 5. Conclusion In the present study, we have observed increased methanogenesis and decreased CH4 oxidation (both low and high affinity) in tropical rice soils in response to elevated CO2 and temperature interaction. Decreased soil redox potential and increased readily available C contents favoring the proliferation of methanogens may be the cause of increased CH4 production at elevated CO2 concentration. At the same time decreased redox potential of soil as a result of CO2 enrichment, resulted in depression of methanotrophic bacterial population leading to decreased methanotrophy in this CO2 enrichment study. However, this finding should be interpreted with caution because these responses were observed during a short-term incubation study under highly controlled conditions. Long-term studies under simulated field situations including rice plants are needed to expand the horizon of our knowledge on the responses of methanogenesis and methanotrophy to elevated CO2 and temperature interaction. Acknowledgments S. Das was supported by a fellowship from the SAC-CRRI Collaborative project, granted to the corresponding author. The manuscript is a part of the Ph. D. dissertation submitted by the senior author to the Utkal University, Bhubaneswar, India. References Adhya, T.K., Mishra, S.R., Rath, A.K., Bharati, K., Mohanty, S.R., Ramakrishnan, B., Rao, V.R., Sethunathan, N., 2000. Methane efflux from rice-based cropping systems under humid tropical conditions of eastern India. Agriculture, Ecosystems and Environment 79, 85e90. Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press, CA. Allen, A.S., Andrew, J.A., Finzi, A.C., 2000. Effects of free-air CO2 enrichment (FACE) on below-ground processes in a loblolly pine (Pinus taeda). Ecological Applications 10, 437e448. Allen Jr., L.H., Albrecht, S.L., Colon-Guasp, W., Covell, W.S.A., Baker, J.T., Pan, D., Boote, K.J., 2003. Methane emissions of rice increased by elevated carbon dioxide and temperature. Journal of Environmental Quality 32, 1978e1991. Ambus, P., Robertson, G.P., 1999. Fluxes of CH4 and N2O in aspen stands grown under ambient and twice-ambient CO2. Plant and Soil 209, 1e8. Bardgett, R.D., Freeman, C., Ostle, N.J., 2008. Microbial contributions to climate change through carbon cycle feedbacks. The ISME Journal, 1e10. Bardgett, R.D., Wardle, D.A., 2010. AbovegroundeBelowground Linkages: Biotic Interactions, Ecosystem Processes and Global Change. Oxford University Press, Oxford, U.K., 1e301 pp.

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