Yao und Conrad - 1999 - Thermodynamics of methane production in different

PERGAMON 

Soil Biology and Biochemistry 31 (1999) 463±473 

Thermodynamics of methane production in di€erent rice paddy 

soils from China, the Philippines and Italy 

Heng Yao, Ralf Conrad * 

Max-Planck-Institut fuÈr terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany 

Received for publication 7 October 1998 

Abstract 

Methane production was measured in anoxic slurries of rice ®eld soils that were collected from 16 di€erent sites in China, the 

Philippines and Italy. The following general pattern was observed. Methane started to increase exponentially right from the 

beginning of anoxic incubation at positive redox potentials (360±510 mV). The concentrations of H 2 and acetate during this ®rst 

phase allowed exergonic methanogenesis with Gibbs free energies of

464 

H. Yao, R. Conrad / Soil Biology and Biochemistry 31 (1999) 463±473 

(Fetzer and Conrad, 1993). Roy et al. (1997) observed 

that Italian rice ®eld soil immediately started to produce 

trace amounts of CH 4 after the soil was ¯ooded. 

Inhibition experiments suggested that this early CH 4 

was produced by H 2 /CO 2 -utilizing methanogens 

which apparently were not inhibited either by the 

high redox potential or by the presence of inorganic 

oxidants such as Fe(III) and sulfate at this early 

stage of ¯ooding. 

To better understand the chain of events that ®nally 

leads to vigorous CH 4 production, we investigated 16 

di€erent rice ®eld soils which were incubated as anoxic 

slurries. We measured the concentrations of reactants 

and products of the H 2 and acetate-dependent methanogenesis 

and calculated the thermodynamic conditions 

for these reactions after onset of anoxic 

conditions. 

2. Materials and methods 

Soil samples were obtained in autumn or winter, 3±4 

months after rice harvest. The soil was broken into 

lumps of 3±4 cm dia, air dried and stored in darkness 

at 48C until experiments were performed. The soils 

have been described by Yao et al. (1998). The main 

characteristics are given in Table 1. The aerobic storage 

of dried soil has been shown to have no signi®cant 

e€ect on soil methane production capacity 

(Mayer and Conrad, 1990). For experiments, soil 

samples were pulverized and passed through stainless 

steel sieves to obtain soil particles between 0.1 and 

1 mm dia. Ten grams of soil and 10 ml of distilled 

water were put into a 120-ml serum bottle. The distilled 

water was previously bubbled with N 2 for 30 min 

to drive out all dissolved O 2 . The serum bottles were 

closed with butyl rubber stoppers and the headspace 

was ¯ushed with N 2 at a rate of 300 ml min 1 for at 

least 20 min. The incubation temperature was 

3020.38C. All measurements were carried out in triplicate. 

One set of bottles (n = 3) was used to follow the 

partial pressures of CH 4 , CO 2 and H 2 . Gas samples 

were taken from the headspace of the bottles, after vigorous 

shaking by hand for about 30 s, using a gastight 

pressure-lock syringe which had been ¯ushed 

with N 2 before each sampling. Analysis of CH 4 and 

CO 2 was performed on a Shimadzu GC 8A gc 

equipped with an FID and a catalytic converter 

(Chrompack, nickat methanizer). The separation column 

was a 80-cm long Porapak Q 60±80 mesh column 

operated at a temperature of 508C. Analysis of H 2 was 

performed on a Trace Analytical RGD2 HgO±Hg 

vapor conversion detector. Hydrogen was separated 

from other gases using a molecular sieve 5A column 

(80±100 mesh, 70 cm length) at 608C (Conrad et al., 

1987). 

Another set of bottles (n = 3) with soil slurries was 

used for determination of dissolved compounds. The 

soil slurries were repeatedly sampled (1±2 ml) by a 

syringe, after heavy shaking of the bottles by hand. 

The pH and E h of the soil slurries were measured 

with a pH-meter and a Pt-electrode (Wissenschaftlich- 

Technische WerkstaÈ tten GmbH, PH-539). E h readings 

were corrected by a reference electrode (210 mV). Iron 

was measured by taking subsamples of 300 ml which 

were then added to 5 ml of 0.5 N HCl and kept for 

more than 2 h at room temperature. For Fe(II) determination, 

50±100 ml of the HCl suspension was added 

to 1 ml Ferrozin solution (0.1% w/w Ferrozin in 

Table 1 

Characteristics of the soils including the amounts of reducible inorganic electron acceptors 

Soil Origin Initial 

pH 

Initial 

E h 

Organic 

carbon (%) 

Total 

nitrogen (%) 

Nitrate 

(mmol g dw 1 ) 

Fe(III) 


Sulfate 


1 Zhenjiang 7.7 460 1.04 0.07 0.16 146 0.85 

2 Changchun 6.0 510 1.68 0.14 0 170 0.48 

3 Guangzhou 5.1 340 1.85 0.13 0 89 0.84 

4 Beiyuan 7.4 360 1.34 0.09 1.41 87 5.18 

5 Jurong 6.3 460 1.15 0.10 0 196 1.16 

6 Shenyaong 6.7 450 1.35 0.07 0.01 163 1.00 

7 Qinghe 7.6 390 0.95 0.08 0.54 76 0.51 

8 Buggalon 5.9 465 1.97 0.16 0.01 153 1.20 

9 Luisiana 5.1 455 1.65 0.16 0.02 420 1.11 

10 Maahas 6.2 460 2.14 0.17 0 311 1.73 

11 Pila 6.8 400 2.62 0.30 1.66 202 1.16 

12 Gapan 6.0 505 1.51 0.12 0.01 277 0.37 

13 Urdaneta 6.7 430 1.07 0.07 0.32 153 0.28 

14 Maligaya 5.8 480 1.39 0.11 0 205 1.54 

15 Pavia 6.1 440 0.81 0.07 0.45 86 0.45 

16 Vercelli 6.0 480 1.55 0.14 3.69 166 2.07

H. Yao, R. Conrad / Soil Biology and Biochemistry 31 (1999) 463±473 465 

Fig. 1. Accumulation of CH 4 and change of H 2 and acetate during anoxic incubation of 16 di€erent rice ®eld soils from di€erent regions. The arrow indicates the time when 80% of the available 

Fe(III) was converted to Fe(II) and sulfate was reduced to concentrations

466 


50 mM N-2-hydroxyl-ethylpiperazin-N 0 -2-ethane sulfonate, 

pH 7), mixed and centrifuged at 14,000 rpm for 

5 min. The supernatant was measured in a spectrophotometer 

(Hitachi U-1100 photometer) at 562 nm. 

Nitrate, nitrite and sulfate were determined after centrifugation 

of aliquots of the soil slurries and ®ltration 

through a 0.2-mm membrane ®lter (Sartorius, Minisart 

RC15). Part of the ®ltrate was stored frozen (208C) 

for fatty acid analysis. Total sulfate (absorbed and dissolved 

sulfate) was determined after 12 h extraction at 

room temperature using 5 ml phosphate mixture 

(15 mM Ca(H 2 PO 4 ) 2 and 8 mM H 3 PO 4 ) (Nelson, 

1982). Nitrate, nitrite and sulfate were analyzed in a 

Sykam ion chromatographic system containing a 

Sykam (LCA, A09) column and a conductivity detector 

serially connected to a UV detector at 218 nm 

(Bak et al., 1991). Na 2 CO 3 /NaHCO 3 (3 mM/1.5 mM) 

was used as the eluent. Dissolved acetate was 

measured using a Sykam HPLC system equipped 

with an Aminex HPX-87G ion exclusion column and 

Fig. 2. Logarithmic plot of the increase of CH 4 during anoxic incubation of 16 di€erent rice ®eld soils.


a refractive index detector (Erma, CR Inc, ERC- 

7512) (KrumboÈ ck and Conrad, 1991). 

Production rates of CO 2 and CH 4 were determined 

by linear regression of the partial pressures in the 

headspace against time for the appropriate phases of 

incubation. Gibbs free energies (DG) were calculated 

from the actual concentrations of reactants and products 

as described before (Conrad et al., 1986; Peters 

and Conrad, 1996) using the following reactions: 

4H 2 …g†‡CO 2 …g† 4CH 4 …g†‡2H 2 O…l† 

DG 0 …308C† ˆ128:7 kJ 

CH 3 COO …aq†‡H ‡ …aq† 4CO 2 …g†‡CH 4 …g† 

DG 0 …308C† ˆ77:3 kJ: 

The standard Gibbs free energies (DG 0 ) were calculated 

from the standard Gibbs free energies of formation 

of the reactants and products which are 

tabulated in Thauer et al. (1977), and then corrected 

to a temperature of 308C using the Van 't Ho€ 

equation and tabulated values of the standard enthalpies 

of formation (Lange, 1979). 

3. Results 

When slurries of rice ®eld soils were incubated 

under anoxic conditions, H 2 partial pressures immediately 

started to increase and reached relatively high 

values, in some soils >150 Pa, within 3±5 d (Fig. 1). 

Acetate usually behaved similarly, but the pattern was 

not as uniform as that of H 2 (Fig. 1). In some soils 

highest acetate concentrations were reached at a later 

time, usually around d 10±20, forming a further maximum. 

Accumulation of CH 4 (assessed on a linear 

scale) started at di€erent times depending on the soil 

tested (Fig. 1). In some soils, linear CH 4 production 

became apparent as early as 2±3 d after the beginning 

of incubation (soil no. 3, 8, 11 and 15), whereas in 

other soils (no. 13 and 14) more than 40 d passed. In 

many but not all cases, the beginning of CH 4 accumulation 

approximately coincided with the end of sulfate 

and iron reduction (Fig. 1). Eventually, CH 4 production 

became constant and H 2 partial pressures and 

acetate concentrations reached a constant value indicating 

steady state conditions. 

Plotting CH 4 accumulation on a logarithmic scale, 

however, revealed a di€erent picture during the early 

phase of CH 4 production (Fig. 2). At this scale it 

became obvious that CH 4 production started right at 

the beginning of anoxic incubation in all soils examined. 

In ®ve soils (no. 3, 8, 11, 15 and 16) this early 

CH 4 production steadily continued until the end of incubation 

and also became apparent on a linear scale 

after a few days (Fig. 1). In all the other soils, however, 

the early CH 4 production came to a halt 3 d later 

(Fig. 2). At this time, the CH 4 partial pressure was still 

low (about 10±100 Pa), since only little CH 4 (about 

Table 2 

End of the halt phase of CH 4 production and of the phases of reduction of sulfate and iron together with the Gibbs free energies of H 2 /CO 2 - 

dependent methanogenesis and rates of total CH 4 production 

End of (d) DG (kJ mol 1 CH 4 ) CH 4 production (mmol g 

dw 1 d 1 ) 

soil halt phase sulfate 

reduction 

(70 5.8 30.5 ± ± ± 

14 36 70 35 3 24.5 28.8 0.14 0.15 

15 0 6 12 27.9 ± 29.8 0.72 0.13 

16 0 14 14 22.4 ± 28.0 0.43 0.18 

± = not applicable, since either no halt phase or no steady state was observed.

468 


0.04±0.42 mmol g dw 1 soil) had been produced. The 

length of the halt of CH 4 production di€ered among 

the di€erent soils and lasted until d 9±36, in soil No. 13 

for even longer (Table 2). The end of the halt phase 

often, but not always, coincided with the end of iron 

reduction (Table 2). Iron reduction was usually ®nished 

later than sulfate reduction except in two soils 

(No. 4 and 14). 

The halt phase was characterized by an increase of 

the Gibbs free energies of H 2 /CO 2 -dependent methanogenesis 

(DG H2 ) to values > 20 kJ mol 1 CH 4 (Fig. 3; 

Tables 2 and 3). In those soils (No. 3, 8, 11, 15 and 

16) which exhibited no halt phase the DG-values 

stayed at values more negative than 19 to 28 mol 1 

CH 4 . The DG-values during the later stages of CH 4 

production, when steady state for production and consumption 

of methanogenic substrates was reached, 

generally showed DG-values more negative than 20 

kJ mol 1 CH 4 (Table 2), on the average a value of 

26.221.8 kJ mol 1 CH 4 (Table 3). 

The DG-values of acetate-dependent methanogenesis 

(DG AC ) during the steady state were similar among the 

di€erent soils (Table 3). The DG AC -values were highest 

(least exergonic) during the steady state; at earlier 

stages of the anoxic incubations, DG AC was even more 

negative (data not shown). 

Fig. 3. Temporal change of the Gibbs free energy (DG) ofH 2 -dependent methanogenesis during anoxic incubation of 16 di€erent rice ®eld soils.


Table 3 

Gibbs free energies (DG; kJ mol 1 CH 4 )ofH 2 /CO 2 -dependent and acetate-dependent methanogenesis during di€erent phases 

of incubation averaged for the di€erent soils 

Methanogenic substrate; phase Average 295% con®dence limit Range 

H 2 /CO 2 ; begin of halt phase 26.2 7.30 38.8 to 10.5 

H 2 /CO 2 ; least negative DG 9.0 3.75 15.3 to 1.2 

H 2 /CO 2 ; end of halt phase 21.5 4.44 31.1 to 13.2 

H 2 /CO 2 ; steady state phase 26.2 1.76 29.9 to 20.4 

Acetate; steady state phase 28.8 1.67 34.9 to 26.2 

The end of the halt phase did not exactly coincide 

with the end of iron reduction, i.e. the time when inorganic 

electron acceptors were depleted. Sometimes the 

halt phase ended earlier, sometimes later. The period 

during which the end of iron reduction lagged behind 

the end of the halt phase positively correlated with the 

maximum rate of CH 4 production but not with the 

amount of reducible iron (Fig. 4). The maximum rate 

of CH 4 production itself correlated only weakly 

(r = 0.276; p>0.05) with the amount of reducible 

iron, but correlated well with the ratio of total nitrogen 

divided by reducible iron (r = 0.724; p < 0.01) 

(for details see Yao et al., 1998). 

4. Discussion 

Our results con®rm and extend the observation by 

Roy et al. (1997) that CH 4 production in rice ®eld 

soils starts right after establishment of anoxic conditions, 

even though oxidants such as ferric iron or 

sulfate have not yet been reduced and the redox potential 

is still high. All rice ®eld soils that were tested 

showed this initial CH 4 production that became visible 

on an exponential scale. By contrast, such an initial 

CH 4 production was not observed in upland soils (forest, 

agricultural, savanna and desert soil) that had no 

history of CH 4 production (Peters and Conrad, 1996). 

In these upland soils, exponential CH 4 production 

started later than in the rice ®eld soils, when most of 

the sulfate and Fe(III) had been reduced. The reason 

for the di€erent behavior is most probably the initial 

population size of the methanogenic bacteria which is 

very small (often

470 


et al., 1993; Joulian et al., 1996; Ueki et al., 1997) so 

that the population is high right at the beginning of 

¯ooding and does not need to further increase during 

submergence. Indeed, methanogenic populations stay 

virtually constant during the season (SchuÈ tz et al., 

1989; Asakawa and Hayano, 1995). Thus, the methanogens 

have only to change from the dormant into the 

active state. In the upland soils, on the other hand, the 

methanogenic populations start at very low numbers 

and have to grow to allow vigorous CH 4 production. 

Indeed, numbers of methanogens in these soils 

increased as soon as CH 4 production started (Peters 

and Conrad, 1996). 

A prerequisite for the early CH 4 production seems 

to be a suciently high H 2 partial pressure that corresponds 

to Gibbs free energies of H 2 -dependent methanogenesis 

of less than approximately 23 kJ mol 1 

CH 4 . These conditions were given in all of the rice 

®eld soils tested right at the beginning of anoxic 

incubation, even in soil no. 13 in which later on no 

CH 4 was produced for more than 70 d. The early CH 4 

production generally came to a halt when the Gibbs 

free energy had increased beyond a certain value (only 

one exception, see below). On the average, the early 

CH 4 production came to a halt when the DG values 

had increased to > 26 kJ mol 1 CH 4 , and CH 4 

production resumed when it had decreased again to 


always permissive, substrate concentration is not a 

likely signal; acetate concentration was never limiting 

in a thermodynamic sense. However, it may have been 

limiting for the kinetics of the resident methanogens. 

The kinetic acetate threshold concentrations are much 

higher for Methanosarcina-type (190±1180 mM) than 

Methanosaeta-type (7±69 mM) methanogens (Jetten 

et al., 1990). The K m values for acetate are also higher 

(Jetten et al., 1990). Clone libraries of extracted DNA 

and bacterial counts show that both types are common 

in Italian rice ®eld soil (Groûkopf et al., 1998). 

Acetate concentrations were generally sucient for 

acetate utilization by Methanosaeta, but may not have 

been sucient for Methanosarcina. Methanosaeta is a 

rather elusive microbe which needs prolonged incubation 

to grow up in culture. Thus, 55±64 weeks were 

needed for most probable number cultures from 

Italian rice ®eld soil in which ®nally acetate-utilizing 

Methanosaeta were detected in numbers of 10 6 gdw 1 

soil, whereas earlier enumerations never detected this 

species (Groûkopf et al., 1998). However, we are presently 

not sure whether Methanosaeta was suciently 

numerous in the soils right from the beginning of 

incubation and what kind of conditions other than 

sucient acetate are required for expression of acetotrophic 

methanogenesis. 

In conclusion, we have demonstrated that CH 4 production 

in rice ®eld soils starts almost right after onset 

of anoxia. Hence, we refute previous claims that redox 

potentials of less than 150 mV are required by the 

methanogens for initiation of CH 4 production in soils 

(see Introduction). However, this early CH 4 production 

came in most soils to a halt as soon as reduction 

of sulfate and Fe(III) started and did not 

resume before these reduction processes were ®nished 

and the redox potential had decreased accordingly. 

Hence, the redox potential may be an indicator for the 

onset of the late CH 4 production and thus may explain 

why CH 4 ¯uxes from wetland rice ®elds are often 

negatively correlated with the redox potentials that are 

measured in the soil (Neue and Roger, 1993; Mishra et 

al., 1997; Yagi, 1997). However, such a correlation is 

no proof for a mechanistic relationship. In fact, some 

soils did not exhibit a temporary halt during the phase 

when sulfate and Fe(III) were reduced and continued 

CH 4 production until the end of incubation. These 

soils (No. 3, 8, 11, 15 and 16) also exhibited relatively 

high maximum CH 4 production rates and exhibited 

them relatively soon after start of the incubation indicating 

that the supply with substrates was not limiting 

for the methanogens. More research is necessary to 

characterize the turnover of organic matter, acetate 

and H 2 in context to sulfate reduction, iron reduction 

and CH 4 production during these phases. 

Neue and Sass (1994) distinguished four di€erent 

patterns of CH 4 production observed in various rice 

®eld soils and illustrated them by logarithmic plots of 

accumulated CH 4 . Interestingly, these four classes of 

soil followed similar patterns as we show in the present 

study. All soil classes exhibited an immediate CH 4 production 

which was visible on a logarithmic scale. After 

this early CH 4 production class I soils required a long 

time for resumption of CH 4 production similar to soil 

No. 13 in our study. Class II soils exhibited a pronounced 

halt phase in between the early CH 4 production 

and the ®nal steady state, such as soil No. 1, 

2, 4, 5, 6, 7, 9, 10, 12 and 14. Class III and IV soils 

exhibited continuous CH 4 production right from the 

beginning throughout the incubation, such as soil No. 

3, 8, 11, 15 and 16. However, Neue and Sass (1994) 

did not provide an interpretation for these di€erent 

CH 4 production patterns. Our results show that the 

patterns can be explained by the energetics of H 2 - 

dependent methanogenesis which are limited by competition 

for H 2 when sulfate and/or iron reduction is 

taking place. 

Acknowledgements 

We thank H.U. Neue and R. Wassmann, X. Zhang, 

G.X. Chen, J. Yang, X.Y. Shen and W.Z. Song, and 

S. Russo for providing soil samples from Philippine, 

Chinese and Italian rice ®elds, respectively. We thank 

S. Ratering, U. JaÈ ckel, S. Schnell for their help during 

the measurement of iron content and other soil characteristics, 

P. Janssen, V. Peters, A. Bollman, T. Henckel 

for providing technical instructions, T. Wind, D. 

KluÈ ber for the valuable discussion, and J. Knecht, G. 

Kutsch for CHN analysis. HY was supported by a fellowship 

of the Alexander-von-Humboldt foundation. 

This study is a contribution to the German BMBF 

program `Klimaschwerpunkt Spurensto€-KreislaÈ ufe'. 

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