Temperature limitation of hydrogen turnover and methanogenesis in anoxic paddy soil

FEMS Microbiology Ecology Published by Elsevier

45 (1987) 281-289

281

FEC 00132

Temperature limitation of hydrogen turnover and methanogenesis in anoxic paddy soil Ralf

Conrad

a, Helmut

Schlitz

b and Monika

Babbel

a

a Uniuersittit Konstonr, Fakultiit ftir Biologic, D-7750 Konstanr and ’ Fraunhofer Institut ftir Atmosphtirische Kreuzeckbahnstr.

19, D-8100 Garmisch-Partenkirchen,

Umweltforschung,

F.R.G.

Received 17 March 1987 Revision received 26 May 1987 Accepted 26 May 1987

Key words:

H, partial pressure; H, turnover; Activation energy: Gibbs HZ-dependent methanogenesis; Methanogenic community

1. SUMMARY The shift of incubation temperature in anoxic paddy soil from 30°C to 15’C resulted in a reversible decrease of the methane production rate and of the H, steady state partial pressure. Only at 30°C but not at 17” C, total CH, production rates were enhanced by the addition of H,, acetate, or cellulose compared to the control. Apparent activation energies which were calculated from the temperature dependence of CH, production were higher in presence than in absence of excess H,. Decrease of temperature caused a decrease of the H, turnover rate constant and of the Gibbs free energy of H,-dependent methanogenesis, and also resulted in a smaller contribution of H, to total methanogenesis. However, HZ-dependent methanogenesis was significantly stimulated by excess H, and slightly inhibited by acetate at low as well as high temperature. The results show that H,producing bacteria were limited by temperature to a greater extent than the methanogens so that the methanogenic microbial community in paddy soil Correspondence to: R. Conrad, Universitlt Konstanz, Fakultli fiir Biologic, P.O. Box 5560, D-7750 Konstanz, F.R.G. 0168-6496/87/$03.50

0 1987 Federation

of European

Microbiological

free energy;

was limited by the supply of H,. At low as well as high temperatures, excess H, apparently enabled part of the methanogenic community to shift from acetate-dependent to H,-dependent CH, production. At low temperature, excess H, had only this effect, but with increasing temperature, excess H, additionally stimulated total methanogenic activity and eventually even growth.

2. INTRODUCTION Methane is an end-product of anaerobic degradation of organic matter. Large quantities of CH, are formed in anoxic aquatic environments such as marsh areas, paddy fields and peat lands, which contribute significantly to the atmospheric CH, budget [l-3]. At the moment, there is great concern about the emission of CH, from these ecosystems, as the atmospheric CH, content is increasing by ca. 1% per year [4-61. In contrast to profundal lake sediments which have a relatively constant temperature regime, the above mentioned methanogenic environments are shallow and thus, exhibit diurnal and seasonal temperature changes which affect the rates of methane Societies

282

production and emission ([3,7,8], Schlitz et al., in preparation). Koyama [9] and Yamane and Sato [lo] studied CH, formation and decomposition of organic substrates in anoxic paddy soil, and observed highest rates at 35-40 o C. Zeikus and Winfrey [ll] showed that the rate of methanogenesis was severely limited by the in situ temperature in sediments of Lake Mendota taken from different depths, and that the methanogenic communities operated optimally at 30-42°C even when taken from depths where temperatures never exceeded 18” C. The same temperature optimum was observed for the conversion of H, and bicarbonate to CH,. Svensson [12] reported on two different methanogenic communities in peat; one, unaffected by H, and using acetate, with a temperature optimum at 20°C; the other one, oxidizing H,, with a temperature optimum at ca. 28°C. CH, production may not only be limited by temperature but also by the availability of the methanogenic substrates [13-151. Besides acetate, H, is the most important methanogenic substrate in freshwater environments and is produced by fermentative bacteria, e.g. by proton-reducing acetogenic bacteria. However, H, must not accumulate as it is inhibitory to the fermentation process for thermodynamic reasons [16-181. The methanogenic bacteria utilize the produced H, and keep it at the low partial pressure that is required by the complex microbial community. Fermentative H, producers and Hz-utilizing methanogens mutually depend on each other and maintain a balanced turnover of H,. However, it is unclear whether the two microbial populations, the fermenters and the methanogens, respond to a similar extent when the temperature changes and thus, it is unclear how the balance between H, production and consumption is maintained. In this study we address two questions: (1) how H, turnover in anoxic methanogenic paddy soil is affected by changes in temperature, and (2) to which extent temperature limitation of CH, production is due to the limitation of the methanogens themselves or to the limitation of the production of methanogenic substrates. Our observations show that temperature limits H, turnover to a greater extent than Hz-dependent methanogenesis

resulting in the limitation of the methanogenic community by available than by temperature.

3. MATERIALS

AND

microbial H, rather

METHODS

3.1. Soil samples The soil was collected from paddy fields of the Italian Rice Research Institute in Vercelli, located in the valley of River PO. The samples were taken during winter time when the flooding water had been drained from the fields, and were stored as dry lumps of ca. 5-10 cm diameter at room temperature. The aerobic storage of dry soil lumps had no significant effect on the soil’s capacity to resume CH, production as soon as anaerobic conditions were re-established. The handling procedure actually imitated the agricultural practice of draining and flooding paddy fields. The characteristics of the soil have been described [19]. The lumps were broken and the dry soil samples were passed through a screen (0.5 mm) immediately before an experiment. 3.2. Methane production and H, steady state partial pressures Serum bottles (120 ml) were filled with 10 g dry weight soil and 10 ml water, closed with a butyl rubber stopper, and flushed with N,. The bottles were incubated at 30” C for 3-5 days. After this time, the colour of the soil had turned from brown to grey, and CH, production had started. The bottles were then pre-incubated over night at the required temperature, flushed with N, until the headspace was free of H, and CH,, and pressurized with N, to a total pressure of 150 kPa. Incubation was continued, and the headspace was analyzed repeatedly for H,, CH, and CO,. The bottles were shaken vigorously for 30 s before gas samples were taken to ensure equilibration between liquid and gas phase. The partial pressures were calculated from the mixing ratios and the total gas pressure which was measured with a needle manometer. The pH of the anoxic soil suspension was determined at the end of the experiment. Substrates were added as sterile, anoxic solutions to a concentration of 5 mM (acetate), 1

283

mg . g ’ d.w. soil (cellulose), bottles with H/CO, (8 : 2).

or by flushing

the

3.3. Activation energies The apparent activation energies of CH, production were from measurements of CH, production rates at incubation temperatures of 7, 15, 20, 25, 30, and 35 ’ C. The apparent activation energy was calculated from linear regression using the logarithmic form of the Arrhenius equation: In P = In A - ( E,/R)(~/T) with P = CH, production rate, A = Arrhenius constant, E, = apparent activation energy, R = gas constant, and T = incubation temperature (K). The regression coefficients (r > 0.99) indicated significant regression of In P to l/T at the 0.1% level. 3.4. Gas analysis Hydrogen was detected by a H, analyzer based on the HgO-to-Hg vapour conversion technique with a detection limit of 0.2 mPa [20,21]. Methane was measured with a gaschromatograph equipped with a flame ionization detector with a detection limit of 0.3 mPa [22]. CO, was analyzed in an infrared analyzer type UNOR (Maihak, Hamburg) with a detection limit of 1 Pa. 3.5. Thermodynamic calculations The Gibbs free energies (AG) of Hz-dependent methanogenesis under the actual incubation conditions were calculated as described by Conrad et al. [18] using a standard Gibbs free energy (AGO’) of - 135.6 kJ/mol CH, [23] and the actual partial pressures of H,, CH, and the concentration of bicarbonate.. The concentration of bicarbonate was calculated from the CO, partial pressure, the pH, the Henry constant and the pK, for CO, [24]. 3.6. Hydrogen turnover rate constant Consumption of dissolved H, was measured in anoxic soil suspensions without a gaseous head space as described previously [25,26]. Sieved soil samples were mixed with water (500 g d.w. soil plus 500 ml H,O) and incubated at 25°C for 3-5 days in Erlenmeyer flasks under a headspace of N, until methane production had started. Then,

incubation was continued for 3 days at 17” C or 30” C. The anoxic soil suspension was anaerobically transfered into a glass syringe (200 ml) avoiding a gaseous headspace and incubated at the indicated temperature. H, was added as aqueous solution, and the decrease of dissolved H, was analyzed after extraction with H,-free air. The H, turnover rate constant (k,) was determined from the logarithmic decrease of H, at limiting H, concentrations (10 PM H2). 3.7. Radioactive tracer experiments Pressure tubes (25 ml) were filled with 6 ml of anoxic soil suspension which was prepared as described above, pre-incubated over night at the indicated temperature, evacuated and gassed with N, or H,. Anoxic water or acetate solution was added. The experiment was initiated by addition of 1 ml of carrier-free NaHi4C0, solution (3.3 X lo6 dpm; 25 nmol). The tubes were incubated in triplicate at the indicated temperature for 24 h. The reaction was stopped by addition of 0.1 ml of 5 N KOH. After gaseous CO, had been absorbed by the alkali (within 3 h), an aliquot (1 ml) of the gaseous headspace was analyzed for CH, concentration, and another aliquot (1 ml) was analyzed for radioactive CH, using the method described by Zehnder et al. [27]. The gas was injected into a scintillation vial fitted with a septum, equilibrated with 20 ml of toluene-based Quickszint 501 (Zinsser, Frankfurt), and counted in a liquid scintillation counter. The efficiency of dissolution of CH, in the scintillation cocktail was 72%, as determined by injecting known amounts of CH, and analyzing the remaining portion after dissolution. The counting efficiency was > 90%, and was routinely determined by external standardization. After opening and flushing the pressure tubes to remove the 14CH4, the radioactivity in the anoxic soil was determined by using Instagel (Packard, Frankfurt). The recovery of total radioactivity was 70-100%.

4. RESULTS Incubation of anoxic sulted in release of H,

paddy soil at 30” C reand CH, (Fig. 1). CH,

284

Table Effect steady

1 of shift of temperature and addition of substrate state H, partial pressure and rate of methanogenesis

Experiment

a

Control (30 o C) Control (17” C) 17°c+300c 17 o C + acetate 17°c+300c + acetate 17 o C + cellulose 17°c+300c + cellulose 17”C+H, 17°c+300c +H, 0

2 I ' 0

20

LO

60

80 Time

100

120

110

. 160

H, partial pressure (Pa)

CH, production (nmol.h-‘.g-’

3.7 + 1.4 1.1 f0.5 3.8 + 2.0 0.6kO.2

36+ 8+ 28+ 12&

3.1 * 1.0 1.0 * 0.5

78+15 8+ 3

24.5 f 8.5 120000

57*11 lo+ 2

120000

74?25

on

d.w.)

4 2 2 3

a The values are meansf SD of triplicate experiments. The bottles were preincubated for 3 days at 17 o C before temperature was shifted and/or substrates were added to a concentration of 5 mM (acetate), 1 rng’g-’ d.w. soil (cellulose), or by flushing the bottles with H,/CO, (8:2). Incubation was continued for 2 days and then, H 2 partial pressures and CH, production rates were measured. At this time, H, had reached a new, more or less constant steady state value, and CH, was produced linearly with incubation time for at least two days.

[hi

Fig. 1. Effect of temperature change on production of CH, and on H, steady state partial pressure. Anoxic paddy soil (10 g d.w. plus 10 ml water) was incubated in serum bottles (120 ml) under a N, atmosphere. H, and CH, were analyzed in gas samples taken from the headspace.

increased linearly with time during the whole incubation period of 7 days due to a constant production rate of CH,. H, increased until a partial pressure of ca. S-10 Pa was reached after 1 day of incubation. The H, partial pressure stayed at this value for the rest of the incubation period indicating that H, production and H, consumption were in steady state. When the incubation temperature was decreased to a lower temperature, the CH, production rate and the H, steady state partial pressure also decreased (Fig. 1). This decrease was reversed when the incubation temperature was increased again. The results sometimes were different with respect to CH, production rate and magnitude of H, steady state partial pressure which varied between 3 and 15 Pa H, (at 30 o C)

with different batches of soil. However, decrease of temperature invariably resulted in a reversible decrease of CH, production and of H, steady state partial pressure. H, partial pressures measured in gas bubbles collected from Italian paddy fields under in-situ conditions with temperatures between 19” C and 27” C were in a range of l-4 Pa [28]. CH, production at 17’ C was not significantly stimulated when the soil was supplemented with excess acetate or Hz/CO, (Table 1). The H, steady state partial pressure at 17” C was only slightly reduced by addition of acetate. As soon as temperature was increased to 30 o C, however, CH, production was stimulated by the substrates, and the H, partial pressure increased to the value of the substrate-free control incubated at 30 o C. The same was observed when cellulose was added to stimulate fermentative production of inethanogenie substrates. Cellulose had no effect at 17°C. At 30” C, however, cellulose enhanced CH, production and also strongly stimulated H, produc-

285 Table 2 0.

1 day

AA

2doys

Apparent activation energies and presence of excess H, a

lncubatlon

Conditions +H

I-

2

Control H 2 /CO,

of CH,

Apparent activation (Arrhenius constant)

production

in absence

energy (kJ. mol- ‘)

1 day incubation

2 days incubation

68.1 (30.7) 79.8 (35.9)

69.7 (31.3) 91.0 (40.7)

a The apparent activation energies were calculated from the data presented in Fig. 2. The Arrhenius constants (nmol CH,.h-‘.g-’ d.w.) are given as logarithms (In A) and are shown in brackets.

)-

I-

)10

20

30

LO

1°C 1 Fig. 2. Influence of temperature and incubation time on CH, production rates. Experimental conditions see Fig. 1. Temperature

tion so that H, partial pressures increased to levels higher than in the unamended control. Similar results were obtained with glucose as substrate (data not shown). Apparent activation energies were calculated from the rates of CH, production at different temperatures (Fig. 2). The temperature-dependent was significantly increase of CH 4 production stimulated by a H, atmosphere resulting in a higher apparent activation energy (Table 2). This observation indicates that H, limitation of CH, production was more pronounced at higher than at lower temperatures. Incubation in presence of excess H, also resulted in a temporal increase of CH, production rates, however, at higher temperatures to a greater extent than at lower ones (Fig. 2). This observation indicates that synthesis of enzymes and/or growth was induced preferably at higher temperatures when excess H, was available causing a further increase of the apparent activation energy (Table 2). A decrease of temperature resulted in a decrease of the H, turnover rate constant (Table 3). The simultaneous decrease of CH, production

indicates that this rate constant was mainly due to the H, consumption activity of the methanogens at the particular incubation temperature. The decrease of temperature also resulted in a decrease of available energy due to decrease of the H, steady state partial pressure. This is documented by the decrease of the Gibbs free energy of H,-dependent methanogenesis (Table 3). Addition of acetate or H, did not stimulate total methane production at 17” C (Table 4). However, the conversion of 14COz to 14CH4 was strongly stimulated by H, and was slightly inhibited by acetate resulting in a significant change of the specific radioactivity of CH, (Table 4). The contribution of HZ-dependent methanogenesis to total methanogenesis apparently

Table 3 H, turnover rate constants and thermodynamics of Ha-dependent methanogenesis at low and high temperature a Incubation

Parameter

17°C H, turnover

3o”c

rate constant

(b-l) CH, production (nmol’h-’ .g-’ d.w.) Hz partial pressure (Pa) CH, partial pressure (Pa) COa partial pressure (kPa) PH AG(kJ.mol-’

temperature

CH,)

11.3 10.4 0.86 950 1.37 7.4 - 20.7

21.2 39.6 8.45 1530 2.14 1.2 - 33.1

a The AG values were calculated for the reaction 4 Ha + HCO; + H+ = CH, + 3 Ha0 using the actual concentrations (pH, bicarbonate) and partial pressures (Ha, CH,) of the reactants at the incubation temperature.

286

Table 4 Effect of temperature

on H,-dependent

methanogenesis

a

(dpm)

Total CH, (nmol)

Specific radioactivity of CH, (dpm/mmol)

17OC no addition 5 mM acetate H 2 headspace

154 105 1415

149 157 150

1.05 kO.04 0.67 i_ 0.07 9.48 + 1.93

3o”c no addition 5 mM acetate H 2 headspace

849 665 3188

384 563 433

2.21 kO.06 1.16+0.11 7.43 + 1.29

Experiment

14CH,

a Anoxic paddy soil (6 ml) was incubated in triplicate in pressure tubes (25 ml) in presence of carrier-free NaH14C0, solution (1 ml; 3.3 X lo6 dpm) for 24 h.

increased dramatically in presence of excess H, and decreased slightly in presence of acetate. The specific radioactivities of CH, show that the contribution of HZ-dependent methanogenesis was higher at 30’ C than at 17 o C, except when H, was present in saturating concentrations (Table 4). This result indicates that HZ-dependent methanogenesis was stimulated by temperature to a greater extent (i.e., higher apparent activation energy) then methanogenesis from acetate or other methane precursors.

5. DISCUSSION Our results indicate that the temperature limitation of methanogenesis is not only due to the temperature limitation of the methanogenic bacteria themselves, but also due to the temperature limitation of those bacteria which produce the methanogenic substrates. This was shown for H, turnover and HZ-dependent methanogenesis in anoxic paddy soil. Low temperatures (15-17 o C) resulted in a low H, turnover rate constant and in a low CH, production rate from H/CO, demonstrating that the activity of the hydrogenotrophic methanogens was reduced by the low temperature. However, CH, production from radioactive CO, was stimulated by addition of H, demonstrating that H,-de-

pendent methanogenesis was limited by H,. This limitation seemed to be due to the low steady state H, partial pressure and consequently, the low Gibbs free energy available for H,-utilizing methanogens, and was most probably due to the low activity of HZ-producing bacteria. It is interesting that H, partial pressure and consequently, the Gibbs free energy at 30 o C was in the same range ( - 26.8 to - 41.6 kJ/mol CH,) as observed for other methanogenic environments under in situ conditions [18], whereas at 17 o C it was slightly below this range. If we assume that the measured H, partial pressure (pH,) is a steady state value due to the simultaneous activity of H, production (P) by fermenting bacteria and H, consumption by methanogenic bacteria (which is equivalent to the H, turnover rate constant k,), its magnitude would be dependent on the temperature dependence of both, P(T) and k,(T): D/T\

PH, = ;,;;; An increase in H, partial pressure may be due to a relative increase in production, but may also be due to a relative decrease in consumption. The simultaneous increase of both, the H, steady state partial pressure and the H, turnover rate constant, indicates that the HZ-producing bacteria were stimulated by increased temperature to a greater extent than the methanogenic bacteria. This was especially the case with easily fermentable substrates such as cellulose or glucose. Recently, we have shown that most of the H,dependent methane production in anoxic environments is due to synthrophic associations in which HZ-producing fermentative bacteria are juxtaposed to HZ-utilizing methanogens [25,26]. The rate of direct H, transfer between syntrophic partners may thus be much larger than the turnover rate of the free H, pool. We have evidence that in paddy soil, too, most of the H, metabolism is taking place by direct transfer within syntrophic associations (unpublished results). However, the HZ-producing bacteria of these associations are most probably stimulated by temperature in a similar way as free-living HZ-producing bacteria. We are presently investigating the possibility

287

whether the temperature-dependent increase of the H, partial pressure is due to release of excess H, from syntrophic methanogenic associations. Although CH, production was limited by substrate availability, addition of H, or acetate was, if at all, only a slight stimulus of total methanogenesis as long as temperature was kept low. Hence, the methanogenic community in total was indeed limited by temperature rather than by substrate or energy availability. However, addition of H, or acetate apparently resulted in a change of the preference for H, or acetate as substrate by part of the methanogenic community while the total activity did not increase significantly. Thus, addition of H, strongly increased the proportion of Hz-dependent methanogenesis, indicating that a large part of the resident methanogenic community had a high spare capacity to metabolize H, as it became available. Addition of acetate, on the other hand, only slightly decreased the proportion of HZ-dependent methanogenesis indicating that only a small part of the resident methanogenic community had the potential to utilize acetate instead of H,. This is a reasonable result, since many of the acetotrophic methanogens so far isolated are able to use H, instead of acetate [29,30], and with some of them, e.g. strains of Methanosarcina, acetate degradation is even inhibited by H, [31,32]. Methanogens which have been isolated with HZ/CO,, on the other hand, generally are unable to use acetate for methanogenesis. At high temperature (30°C) too, addition of substrate caused a shift of the substrate utilization pattern of the methanogenic populations in a similar manner as at low temperature. Addition of H, resulted in a similar increase of the proportion of HZ-dependent methanogenesis as at low temperature indicating that the spare capacity of the methanogenic community for HZ-dependent methanogenesis was now completely saturated irrespectively of temperature. Even with H,, without addition of H,, the proportion of H,-dependent methanogenesis was slightly higher at high than at low temperature, indicating that the hydrogenotrophic methanogens profited from the better availability of H, due to the higher H, steady state concentration and/or a higher rate of

H, production by juxtaposed syntrophic fermenting bacteria. The preferential stimulation of HZ-dependent methanogenesis by increasing temperature is consistent with the observation that the apparent activation energy of CH, production was higher in presence than in absence of excess H,. The apparent activation energy provides a quantification over all the individual temperature-dependent reactions which contribute to the CH, production. For an individual reaction, the activation energy is an intrinsic property. For its measurement it is necessary, however, that the reaction is only limited by temperature and not by substrate. The increase of the apparent activation energy in presence of excess H, indicates that the potential activity of the resident methanogens was limited by H, rather than by temperature. At high temperature, however, addition of H, or acetate did not only result in a change of the relative contribution of hydrogenotrophic or acetotrophic methanogenic bacteria, but also stimulated the total activity of the methanogenic community. Since the extent of this stimulation increased with incubation time, the addition of substrate at high temperature may have induced enzyme synthesis or growth in the methanogenic community. This may also have resulted in higher apparent activation energies (or Q,, values) in presence (80-90 kJ . mol-‘; ca. 2.7-3.4 Q,,) than in absence of excess H, (68-70 kJ . mol-‘; ca. 2.5

Q,o>. Our results demonstrate that temperature can have a very complex effect on a biogeochemical reaction that is based on a food chain involving many different microbial species. These species may have different temperature optima as shown for methanogenesis in peat [12,33] or in an anaerobic digestor [34], and for nitrate reduction in saltmarsh sediments [35]. In addition to different temperature optima, the different species may also have different temperature characteristics, such as Q,, or apparent activation energy. Our results indicate, that apparent activation energies may in addition be dependent on the availabiity of substrate. Because of the variability of these factors within natural environments it is not surprising that Kelly and Chynoweth [36] observed Q,,

288

values for CH, production varying between 0.83 and 5.7 for the same sediment when sampled at different depths and dates. A similar range was observed during continuous measurements of CH, emission rates in Italian paddy fields that showed diurnal changes parallel to soil temperature with apparent activation energies ranging between 20 and > 300 kJ/mol (ca. 0.8-6.5 Q,,) for different dates (Schiitz et al., in preparation). Quantification of the temperature dependence of methanogenesis and CH, emission and modelling of the mechanisms involved under in situ conditions will be of great importance for estimates of budgets of regional and global biogeochemical CH, cycles.

ACKNOWLEDGEMENTS We thank the Istituto Sperimentale per la Risicoltura (Dr. S. Russo) for the soil samples. This work was financially supported by a grant from the Deutsche Forschungsgemeinschaft (Schwerpunkt Methanogene Bakterien).

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