Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

FEMS MicrobiologyEcology62 (1989) 265-274 Published by Elsevier

265

FEC 00213

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil R a l f C o n r a d , H a n s - P e t e r M a y e r a n d M o n i k a Wi~st Unioersitiit Konstanz, Fakultiit fi~r Biologie, Konstanz, F.R.G.

Received 21 October 1988 Revision received 5 December 1988 Accepted 6 December 1988 Key words: H 2 concentration; H 2 turnover; Interspecies H 2 transfer; Homoacetogenic bacteria; Fermenting bacteria; Rice

1. SUMMARY Interspecies H 2 transfer within methanogenic bacterial associations (MBA) accounted for 95-97% of the conversion of 14CO2 to a4CH4 in anoxic paddy soil. Only 3-5% of the 14CH4 were produced from the turnover of dissolved H 2. The H2-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H2-dependent methanogenesis and the turnover of dissolved H 2 did not change significantly for up to 7 months of incubation. However, while the rates of H2-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H2-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 3 0 ° C to 17 °C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization

Correspondence to: Ralf Conrad, Universit~it Konstanz,

Fakultat fiir Biologie,P.O. Box 5560, D-7750 Konstanz, F.R.G.

of dissolved H 2 by chloroform-insensitive nonmethanogenic bacteria, most probably homoacetogens, so that C H 4 production was almost completely restricted to H2-syntrophic MBA. The activity of MBA, as measured by the conversion of 14CO2 to 14CH4, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous HE. Propionate and acetate had no effect.

2. I N T R O D U C T I O N Submerged paddy fields are one of the most important sources in the global budget of atmospheric CH 4 [1]. About 20-50% of this methane seems to be produced from hydrogen as methanogenic substrate [2-4]. Recently, we have shown that more than 95% of the HE-dependent CH 4 production in anoxic paddy soil is due to interspecies H E transfer (ISHT) between juxtaposed HE-producing fermenting and HE-COnsuming methanogenic bacteria [3]. A similar importance of such He-syntrophic methanogenic bacterial associations (MBA) has been demonstrated for other

0168-6496/89/$03.50 © 1989 Federation of European MicrobiologicalSocieties

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methanogenic environments, e.g. anaerobic digestors [5,6] and anaerobic lake sediments [5,7]. In Lake Mendota sediments, the contribution of H2-syntrophic MBA to total C H 4 production apparently increased during the season [7]. The onset of stratification in spring and the formation of an anoxic hypolimnion may be the prerequisite for an increasing number of fermenting and methanogenic bacteria to form H2-syntrophic MBA until this process is stopped during fall turnover. In rice paddy fields, soil and vegetation conditions also change with season, and affect C H 4 emission fluxes [4,8,9]. The most dramatic changes for soil microbial communities take place after flooding of the paddy fields when the relatively dry and oxic paddy soil gets submerged and anoxic, and after shooting of the rice plants when root exudates add to the supply of organic matter for the methanogenic bacterial community [4,9]. We studied the gas metabolism of submerged paddy soil to find out whether prolonged submergence of paddy soil, presence of rice plants, and temperature may have an influence on the contribution of Hz-syntrophic MBA to methane production. Our results indicate that H2-syntrophic MBA were formed at a very early stage after onset of anaerobiosis and increasingly contributed to total methanogenesis owing to decreasing contribution of methanogenic substrates other than H 2.

3. M A T E R I A L S A N D M E T H O D S

3.1. Preparation of anoxic paddy soil The soil was collected from paddy fields of the Italian Rice Research Institute in Vercelli. Storage and handling was done as previously described [10]. Soil slurries were prepared by adding 1 ml of water to each 1 g of dry weight soil. The slurries were incubated under an atmosphere of N 2 / C O 2 (8:2). After 3 days the colour of the soil turned from brown to grey, and C H 4 production started. After various times of anaerobic incubation at constant temperature, aliquots of anoxic soil slurries were transferred into serum bottles to measure C H 4 production rates, H 2 steady state partial pressures, and conversion of 14CO2 t o 14CH4; or

were transferred into glass syringes to measure H2 turnover rate constants.

3.2. Cultivation of rice in the laboratory Paddy cultures were set up as described earlier [8]. Briefly, glass beakers (800 ml) were filled with 600 g d.w. soil and then flooded with distilled water so that the soil surface was covered by about 2 - 5 cm of water. About 10 rice seeds (Oryza sativa var. Roma, type japonica) were planted in each beaker and incubated at 2 0 ° C under illumination. The rice cultures were used to measure C H 4 emission rates using the incubation apparatus described previously [8]. Anoxic paddy soil was then harvested from the rice cultures using a glove bag filled with N 2 / C O 2 ( 8 : 2 ) and was diluted with anoxic distilled water to give the water content ( < 1 ml H 2 0 per 1 g d.w. soil) desired for further experiments. Water content was determined gravimetrically after drying at 104°C. The soil slurries were then used to determine total C H 4 production, 1 4 C H 4 production from 14CO2, steady state H 2 partial pressures, and H 2 turnover rate constants. 3.3. Methane production and H 2 steady state partial pressures Pressure tubes (25 ml) or serum bottles (60 or 120 ml) were filled with 10-20 ml of anoxic soil slurry, closed with a butyl rubber stopper, flushed with N2, and pressurized to a total pressure of 150 kPa. The following days, the headspace was repeatedly analyzed for H 2 and C H 4. The bottles were shaken vigorously for 30 s before gas samples were taken to ensure equilibration between liquid and gas phase. C H 4 increased linearly with time, and H 2 partial pressures reached steady state values after 1 - 2 days of incubation. Dissolved H 2 concentrations (nM) were calculated from the partial pressure and the Bunsen solubility coefficient [11] at the incubation temperature, and were corrected via the water content of the soil slurry to give the H 2 concentration (CH2) on a gram d.w. basis of soil. 3.4. Hydrogen turnover rate The anoxic soil slurry was transferred anaerobically into a glass syringe (200 ml) avoiding a

267

gaseous headspace, and incubated at the desired temperature. H2 was added as aqueous solution, and the decrease of dissolved H 2 was analyzed after extraction with H2-free air as described previously [5,10]. The H 2 turnover rate constant (kH2) was determined from the logarithmic decrease of H : at limiting H 2 concentrations (10 /~M). In steady state, the H 2 production rate (PH2) which is equivalent to the H 2 turnover rate was calculated from its turnover rate constant and the steady state concentration of dissolved H 2. To calculate the percentage (fCH4) of H 2 turnover due to H 2oxidizing methanogenic bacteria, H 2 turnover rate constants ( k i n ) were also measured in the presence of 100 /~M chloroform which completely inhibited methane production.

3. 6. Production of H e and interspecies H 2 transfer (ISHT) Production rates of H2/CO2-dependent methanogenesis, i.e. production of 14CH4 from 14CO2, were calculated from total C H 4 production rates times fn2. H2 production rates necessary to sustain production of 14C H 4 were calculated from the stoichiometry 4 H 2 = 1 c n 4. For steady state conditions, turnover of dissolved H2 is equivalent to production and consumption of dissolved H 2. Only part of the total turnover of dissolved H e is used by methanogens. The use of H 2 for 14CH4 production is balanced by the consumption of dissolved H 2 and by the rate of ISHT within H2-syntrophic methanogenic bacterial associations (MBA): 4PcH4fH2 = ISHT + kH2CH2fCH4

3.5. Fraction of H 2/ C02-dependent methanogenesis Pressure tubes (25 ml) or serum bottles (60 or 120 ml) were filled with 20 ml Of anoxic soil slurry, closed with butyl rubber stoppers, evacuated and gassed with N 2. The experiment was initiated by addition of 100/~1 of carrier-free NaH14CO3 solution (6.6 × 106 dpm; 50 nmol). The bottles were incubated in duplicate at the desired incubation temperature. During the next two days, the headspace was repeatedly analyzed for total CH4, CO 2 and H 2, as well as for radioactive 14CH4 and 14COz. The bottles were shaken vigorously for 30 s before gas samples were taken to ensure equilibration between liquid and gas phase. Total CH 4 and CO 2 (#mol m1-1 of headspace) as well as 14CH4 and 14CO2 (dpm ml-a of headspace) increased linearly with time giving a constant specific radioactivity of C H 4 and CO 2 over the incubation period of 48 h. The fraction of H2/CO2-dependent methanogenesis was calculated from the specific radioactivities of C H 4 and CO2: fH2 ----SRcH4/SRco2

where fH2 (no dimension) is the fraction of H2/CO2-dependent methanogenesis, SRcH 4 (dpm /~mo1-1) is the specific radioactivity of CH4, and SR co2 (dpm/~ m o l - 1) is the specific radioactivity of CO 2.

ISHT(%) = (4PcH4fH2 -- kH2fHzfcrt4 ) X (100/4PcH4 f H2 )

where ISHT (nmol h -1 g-1 d.w.) is the rate of H 2 transfer within H2-syntrophic MBA, PCH4 (nmol h-1 g - i d.w.) is the rate of total CH4 production, fn2 (no dimension) is the fraction of H2/CO2-dependent methanogenesis, k m (h -1) is the H 2 turnover rate constant, C H2 (nmol g-1 d.w.) is the steady state H 2 concentration, and fCH4 (no dimension) is the fraction of H e turnover due to methanogens (fraction of k H2 that is sensitive to chloroform inhibition).

3. 7. Gas analysis Analysis of H2, C H 4 and CO e was done as described previously [10]. 14CH4 and 14CO2 were analyzed in a gas chromatograph at 50 ° C using a Porapak QS (80/100 mesh) separation column (length = 3 m; i.d. = 4 mm) and H 2 as carrier gas at a flow rate of 25 ml min -~. Behind the separation column the gas stream was passed through a methanizator (Ni-Catalyst at 330°C; Chrompak, Middelburg, Netherlands). The gas stream was then split and fed into a flame ionisation detector (Carlo Erba, Milano, Italy) and a RAGA radioactivity detector for gas chromatography (Raytest, Straubenhardt, FRG). The radioactivity signal was calibrated by using 14CO2 which was prepared from NaH14CO3 standard solutions.

268

4. RESULTS 4.1. Initial phase of anoxic soil conditions When air-dried paddy soil was suspended in anoxic water and incubated at 3 0 ° C under an O2-free headspace of N 2, the colour of the soil turned from brown to grey, the reducing potential decreased from + 180 mV to - 2 3 0 mV, and CH 4 production started within two days. H 2 was produced during the first day and reached partial pressures of 500 Pa (3.3/~M) (Fig. 1). Subsequently, it decreased again and after 3 days of incubation reached a value of about 4 Pa (26 nM) (Fig. 1) which is typical for steady state conditions at 3 0 ° C [10]. H 2 turnover rate constants could be measured already after 10 h incubation, but were insensitive to inhibition by chloroform until the 2nd day of incubation. During the initial 2 days H 2 apparently was used by bacteria other than methanogens, probably by chemolithotrophic homoacetogenic bacteria (in preparation). During the second day, CH 4 started to increase and H 2 turnover rate constants due to methanogenesis (i.e., kH2fCH4) increased simultaneously

(Fig. 1). The percentage (fH2) of C H 4 produced from H 2 a n d 14CO2 increased as well and reached 6% on the fourth day (Fig. 1). On the fourth day, turnover of dissolved H 2 reached steady state conditions and thus, could be compared to the rate of H 2 consumption required for conversion of 14CO2 to 14CH4. The calculations showed that 85% of H2/CO2-dependent methanogenesis was due to interspecies H 2 transfer (ISHT) within H2-syntrophic methanogenic bacterial associations (MBA). This percentage further increased to 95% ISHT within the next 3 days. Unfortunately, the percentage contribution of ISHT to H2/CO2-dependent methanogenesis could not be calculated during the first 3 days of incubation since H 2 production and consumption reactions obviously were not yet in steady state. However, we assume that H2-syntrophic MBA started their activity during this initial phase. 4.2. Gas metabolism in anoxic soil slurries Prolonged incubation of anoxic paddy soil for up to 33 days resulted in a gradual decrease of total C H 4 production rates, but in an increase of the contribution of H2/CO2-dependent methano-

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Fig. 1. Temporal change of 8 2 and CH 4 metabolism during the initial phase of anoxic incubation at 30 o C of submerged paddy soil. Air-dried soil was suspended in O2-free water and incubated under a nitrogen atmosphere in several replicates. Three replicates were analyzed at each time point for H 2 and CH 4. Three replicates were amended with NaH14CO3, and their headspace was analyzed for 14CH4 and 14CO2 to determine the value of fH2" Two replicates were opened at each time point to measure the H 2 turnover constant (k H2) in the presence and absence of chloroform to calculate the value of k H2 fCH4"

269 Table 1 Temporal change of gas metabolism and interspecies hydrogen transfer (ISHT) in H2-syntrophic methanogenic bacterial associations (MBA) after onset of anoxic conditions in paddy soil slurries incubated at 25 o C a Kinetic parameter

Pcn4, total CH 4 (nmolh -1 g-ld.w.) fn2 (%) 14CH4 from 14CO2 (pmol h -1 g-1 d.w.) H E used for 14CH4 (pmol h -1 g-1 d.w.) kH2 ( h - 1) CH2 (nM = pmol g-1 d.w.) H 2 production (pmol h -1 g - I d.w.) fCH4, H2 consumed by methanogens (%) ISHT (%)

Period of anoxic incubation (days) 5

25

33

44.1+5.5 6.9±2.4

22.4±6.0 14.6+2.1

21.9±5.3 17.2±1.2

2999

3270

3767

11996 21.8

13080 22.7

15068 18.1

28.8

30.6+3.8

34.0+4.2

628

695

615

38 97.1

69 96.3

66 97.3

tively low values of about 10 nmol h -1 g-1 d.w. (Table 2). CH 4 production rates were slightly higher when the soil had been planted with rice. Only part of the produced CH 4 was emitted from the cultures into the atmosphere (Table 2). Contribution of H2/CO2-dependent CH4 production to total methanogenesis was similar, but production of dissolved H 2 was much higher in rice-planted than in unplanted soil (Table 2). On the other hand, most of the dissolved H 2 was not used by methanogens since H 2 turnover rate constants were only partially sensitive to inhibition by chloroform. H 2 consumption by non-methanogens was higher in rice-planted than in unplanted soil (Table 2). Anyway, turnover rates of dissolved H e were significantly lower than rates of H 2 used for conversion of 14CO2 to 14CH4 so that most of it must have been produced by ISHT within H2-syntrophic MBA contributing about 95-99% to H2/CO2-dependent methanogenesis (Table 2).

a Mean values ± SD.

genesis (Table 1). However, the total amount of n 2 used for production of 1 4 C H 4 increased only slightly because of the decreasing rate of total CH 4 production. H 2 turnover rate constants and steady state concentrations of dissolved H 2 stayed more or less constant during the entire incubation period resuiting in constant rates of production of dissolved H 2. However, these rates were about 200 times lower than required for production o f 14CH4 from 14CO2 indicating that most of the H2/CO2dependent methanogenesis took place by ISHT within H2-syntrophic MBA accounting for about 95% of C H 4 production from H 2. Since a significant part of dissolved H 2 was used apparently by chloroform-insensitive non-methanogenic bacteria, contribution of ISHT within H2-syntrophic MBA actually amounted to 96-97% (Table 1) during the entire incubation period of about one month. 4. 3. Gas metabolism in rice cultures To mimic field conditions, anoxic paddy soil was prepared by submersion in beakers under ambient air for 3 to 7 months. After this period, total CH 4 production rates had decreased to rela-

4. 4. Effect of incubation temperature on I S H T Incubation of anoxic soil slurries at 30 °C generally resulted in higher rates of total C H 4 proTable 2 H2-dependent methanogenesis in planted and unplanted paddy cultures incubated at 20 ° C a Parameter

rice

unplanted

unplanted

Months incubated H 2 0 (ml g - i d.w.) PCH4, total CH 4 (nmol h - 1 g - I d.w.) CH 4 emission (nmol h - 1 g - 1 d.w.) CH 4 oxidized (%)

3-4 0.63

3-4 0.67

6-7 0.86

13.1±1.0

8.5___3.7

7.0±0.4

2.2 83

6.7 21

filE (%)

19.1±1.4

15.9±3.3

24.5±1.5

2173

1351

1715

8692 58.7+1.9 59.0+11.9 37.2

5404

6860 36.9±6.0 19.5+1.7 16.8

14CH4 from 14CO 2 (pmolh -1 g-ld.w.) H 2 used for 14CH4 (pmol h -1 g - i d.w.) kH2 (h -1) CH2 (nM) CH2 (pmol g - i d.w.) H 2 production (pmol h -1 g - i d.w.) FcH4, H 2 consumed by methanogens (%) ISHT (%) a Mean values + SD.

27.7±5.2 18.5

2178

620

5 98.7

46 95.8

270 Table 3

Table 4

Temporal change of methane production from H 2 / C O 2 in anoxic paddy soil before and after shift of incubation temperature from 30 ° C to 17 ° C a

Influence of temperature on rates of H 2 turnover and methanogenesis in anoxic paddy soil a

Kinetic parameter

5 30 ° C: P¢~4, total C H 4 (nmol h - 1 g - 1 d.w.) fH2 (%) 14CH4 from 14CO2 (pmol h -1 g - 1 d.w.) H 2 used for 14CH4 (pmol h -1 g - 1 d.w.) 17°C: Penn, total C H 4 (nmol h -~ g - I d.w.) fH2 (%) 14CH4 from 14CO2 ( p m o l h -1 g ad.w.) H 2 used for 14CH4 (pmol h -1 g - 1 d.w.)

Incubation temperature

Parameter

Period of anoxic incubation (days) 7

18

67 ± 2 4.9±0.4

49 + 8 7.8___0.4

32 ± 4 30.5±4.5

3283

3822

9760

13132

15288

39040

26±1 2.0+_0.2

16±1 4.8±1.0

13±1 21.2+_2.8

520

768

2756

2080

3072

11024

a Anoxic paddy soil (1 g d . w . + l ml H 2 0 ) was incubated at 30 ° C. One day before the time indicated the soil slurries were preincubated at 17 or 30 ° C, then the measurements were initiated; mean values + SD.

duct±on than incubation at 25 ° C (Tables 1 and 3). Furthermore, the contribution of H2/CO2-dependent C H 4 production (fH2) at 3 0 ° C increased relatively faster with incubation time and reached relatively higher values (Tables 1 and 3). However, the fH2-values decreased within one day after the temperature was changed from 3 0 ° C to 1 7 ° C (Table 3). The relative decrease was higher at low than at high values of fH2, i.e. it was higher after 5 days than after 18 days of anoxic incubation. The temperature-dependent decrease of both, fH2 and total methanogenesis, resulted in decreased rates of H 2 used for methanogenic conversion o f 14CO2 t o l n C H a (Table 3 and 4), e.g., shifting the temperature after 6 days of incubation at 3 0 ° C to 1 7 ° C resulted in a decrease of the H 2 requirement for production o f 14CH4 to 20% of its original value (Table 4). Simultaneously, the steady state concentration of dissolved H a and its turnover rate constant decreased as well and resuited in a decrease of the production rate of dissolved H 2 to 4% of its original value (Table 4).

PCH4, total C H 4 (nmol h - 1 g - 1 d.w.) fH2 (%) 14CH4 from 14CO2 (pmol h -1 g - 1 d.w.) H 2 used for 14CH4 ( p m o l h l g ld.w. ) kH2 (h -1 ) C H2 (nM) H 2 production (pmol h - 1 g - 1 d.w.) fcH4, H2 consumed by methanogens (%) ISHT (%)

30°C

17°C

49 7.8

16 4.8

3822

768

15288 43 24

3072 33 1.5

1032

49.5

65 95.6

33 99.5

a Anoxic paddy soil (1 g d . w . + l ml H z O ) was incubated for six days at 30 o C, then for one day at 17 or 30 ° C, then the measurements were initiated; mean values ± SD.

After the temperature shift to 1 7 ° C the turnover of dissolved H 2 apparently contributed less to H2-dependent methanogenesis than before, indicating that I S H T within H 2 syntrophic MBA became more important. This is even more the case if we consider that the turnover of dissolved H 2

Table 5 Influence of organic electron donors on H 2 partial pressure, total C H 4 production rates and H 2 / C O 2 - d e p e n d e n t methanogenesis in anoxic paddy soil a Substrate (10 m M )

H : partial pressure (Pa)

Control H2 Glucose Ethanol Lactate Propionate Acetate

0.9+ 12000 78.7 ± 17.7± 7.6± 0.4± 1.2±

a

0.5 25.5 9.9 4.6 0.2 0.1

CH 4 production rate, PCH4 (nmol h - 1 g i d.w.)

fH2 (%)

31+- 4 36_+ 1 51 ± 1 65±13 39± l 21± 8 51+_15

7+ 1 24+ 1 35_+ 8 25±10 20_+ 5 5± 3 3_+ 2

Anoxic paddy soil (6 ml) was incubated in pressure tubes (25 ml) in presence of organic electron donors and carrier-free NaH14CO3 solution (3/~Ci) at 25 ° C for 2 days. The figures are m e a n values + SD of triplicate measurements.

271 was only partially due to methanogens and that methanogens contributed less at low than at higher temperature (Table 4).

4.5. Stimulation of He/COe-dependent methanogenesis by organic substrates Addition of glucose, ethanol or lactate, but not of propionate stimulated the rates of total and even more those of H2/COz-dependent CH 4 production (Table 5). The stimulation by these substrates was higher than that by H 2, although the resulting H 2 partial pressures were much lower. Acetate also stimulated total methanogenesis but the contribution of H2/CO2-dependent C H 4 production was decreased (Table 5).

5. DISCUSSION Our results indicate that hydrogen-dependent methanogenesis in anoxic paddy soil was predominantly due to H2-syntrophic methanogenic bacterial associations (MBA). The MBA converted 14CO2 t o 1 4 C H 4 by using reducing equivalents which were not provided from the pool of dissolved H 2 but apparently came from within the MBA. The MBA were most probably formed by H2-producing fermenting bacteria and H2-consuming methanogenic bacteria that allow interspecies H 2 transfer (ISHT) between juxtaposed cells. These H2-syntrophic MBA were relatively stable to dilution [10] and started activity briefly after submerging the paddy soil and imposing anoxic conditions. They apparently were fully developed after only 5 days of incubation under anoxic conditions. The MBA may have formed from individual fermenting and methanogenic bacteria as soon as conditions became anoxic and sufficiently favourable for growth. Alternatively, the MBA may have survived in the dry and oxic paddy soil and just have revitalized when the soil was submerged again. We actually favour the second explanation, since MPN counts of hydrogenotrophic methanogenic bacteria were in a range of 106 counts g-1 d.w. irrespective of whether the tested soil was submerged, anoxic, and methanogenic, or was dry, oxic, and inactive (Mayer and Conrad, in

preparation). Even under field conditions, MPN counts of hydrogenotrophic and acetotrophic methanogens give a constant value of 10 6 and 10 4 g-1 d.w., respectively, throughout the growing season [4]. The contribution of H2-syntrophic MBA to H2-dependent methanogenesis was about 95-97%, the rest being due to methanogenic consumption of the dissolved H 2 pool. The contribution of H2-syntrophic MBA did not change significantly during prolonged incubation under anoxic conditions, and was similar in slurries of paddy soil and in laboratory paddy cultures mimicking field conditions. Similarly, the production rates of dissolved H 2 stayed almost constant for incubation periods of 5 days to up to 7 months. The situation in paddy soil therefore seems to be different from the situation in stratified Lake Mendota sediments where the contribution of MBA increased during the season [7]. Despite the relatively constant contribution of MBA to H2-dependent methanogenesis, gas metabolism changed significantly during anoxic incubation of paddy soil, and was also significantly affected by the presence of rice plants. Total methanogenesis decreased during prolonged incubation, probably because of depletion of organic substrates, but was enhanced in the presence of rice plants, probably because of supply of additional organic substrates by root exudation a n d / o r autolysis [9]. The rhizosphere of rice plants also provided a more efficient environment for reoxidation of the produced CH4 than the shallow oxic layer at the paddy soil surface [8,9]. The rate of H 2 used for conversion of 14CO2 to 14CH4 increased only slightly during the first month of incubation. Nevertheless, the contribution of H2-dependent methanogenesis (95-97% by H2-syntrophic MBA) to total C H 4 production increased significantly from about 5% to 25% during this time. This increase of contribution was mainly due to the decrease of the rates of total methanogenesis rather than to the slight increase of the rates of H2-dependent methanogenesis. Obviously, methanogenic substrates other than H2 were gradually depleted so that the contribution of H2/CO2-dependent methanogenesis increased. During the next 2-6 months of incubation, this

272 contribution stayed in a range of 15-25% and reached only slightly higher values (30%) during incubation at 30°C. Somewhat higher values (30-50%) were measured in freshly sampled soil cores from the paddy field in Italy [4]. Although the contribution of H/-syntrophic MBA to H2-dependent methanogenesis was high at 95-97%, it even further increased to > 99% when the incubation temperature was decreased. Two effects could possibly be involved: (1) Decreased temperature results in decreased rates of H 2 production by fermenting bacteria, both freeliving and MBA-associated [10]. (2) Decreased temperature also results in competitive utilization of dissolved H E by psychrotrophic chemolithotrophic homoacetogenic bacteria [12]. Competition by chemolithotrophic homoacetogens should affect only the free-living methanogens but not the HE-syntrophic MBA. It is unknown, however, whether HE-syntrophic homoacetogenic bacterial associations exist in addition to HE-syntrophic MBA. We are presently conducting experiments to determine the incorporation of 14CO2 into the acetate pool. Preliminary results exclude the possibility that HE-dependent C H 4 formation is mimicked by acetoclastic C H 4 production from the resulting a4C-labelled acetate. An increase to 99% of the contribution of H 2syntrophic MBA to HE-dependent methanogenesis was also observed in soil planted with rice. Although the presence of rice plants stimulated the production of dissolved H E, it was mainly used by homoacetogens rather than by methanogens. The dissolved H E pool generally was shared to a large extent by homoacetogenic bacteria, especially at low temperatures [12]. However, their contribution was especially large in the presence of rice plants, and reached a value of 95%. It is unknown how homoacetogens can successfully compete with methanogens for dissolved H 2 under these conditions. This situation is similar to the hindgut of termites, where homoacetogens also outcompete methanogens despite less favourable kinetics and tresholds for dissolved H 2 [13,14]. The bacterial composition and metabolism of MBA have so far only been a n a l y z e d using anaerobic digestor flocs and enrichment cultures [6,15-18]. The species composition of the HE-syn-

trophic MBA from anoxic paddy soil is not yet known. Mesotrophic Methanobacterium bryantiilike bacteria have been isolated from paddy soils as dominant HE-COnsuming methanogens [12,19]. Sulfate-reducing bacteria which ferment ethanol and lactate to acetate, CO z, and H E apparently were the predominant HE-producing bacteria in HE-syntrophic MBA from whey digestor flocs [6,18] and from Lake Mendota sediment [7]. In other MBA-containing systems, butyrate-degrading clostridia [17] or propionate-degrading Syntrophobacter sp. seemed to be the main HE-producing bacteria. In MBA from anoxic paddy soil, the HE-producing fermenting bacteria are not yet known. Our results indicate, however, that glucose, lactate, and ethanol-fermenting bacteria are more likely candidates than propionate-fermenting bacteria.

ACKNOWLEDGEMENTS We thank the Rice Research Institute (Dr. Russo) in Vercelli, Italy, for provision of soil samples and rice seeds. This work was financially supported by a grant from the Deutsche Forschungsgemeinschaft (Schwerpunkt Methanogene Bakterien).

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