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

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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)

(mmol g dw 1 )

Sulfate

(mmol g dw 1 )

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


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

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.


H. Yao, R. Conrad / Soil Biology and Biochemistry 31 (1999) 463±473 467

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

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

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.


H. Yao, R. Conrad / Soil Biology and Biochemistry 31 (1999) 463±473 469

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


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

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


H. Yao, R. Conrad / Soil Biology and Biochemistry 31 (1999) 463±473 471

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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