APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1997, p. 4552–4557 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 63, No. 11
Inhibition of Methanogenesis by Methyl Fluoride: Studies of Pure and Defined Mixed Cultures of Anaerobic Bacteria and Archaea PETER H. JANSSEN*
AND
PETER FRENZEL
Max-Planck-Institut fu ¨r Terrestrische Mikrobiologie, D-35043 Marburg, Germany Received 2 May 1997/Accepted 26 August 1997
Methyl fluoride (fluoromethane [CH3F]) has been used as a selective inhibitor of CH4 oxidation by aerobic methanotrophic bacteria in studies of CH4 emission from natural systems. In such studies, CH3F also diffuses into the anaerobic zones where CH4 is produced. The effects of CH3F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH3F, similar to the amounts used in inhibition experiments, inhibited growth of and CH4 production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH3F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH3F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH3F of the growth and CH4 production of Methanosarcina mazei growing on acetate was reversible. CH3F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH3F as a selective inhibitor of aerobic CH4 oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH3F may be useful for the rapid determination of the role of acetate as a CH4 precursor. The contribution of rising CH4 levels in the Earth’s atmosphere to the greenhouse effect (31) has prompted increased research into the identification and quantification of the sources and sinks of CH4 in the biosphere (12, 21). Methanogenic archaea are the biological sources of CH4 and are found in a wide range of anoxic environments. They utilize the end products of fermentative degradation of organic matter, hydrogen and acetate, in energy-yielding reactions which allow them to grow and which produce CH4 as the end product. Among the dominating sources of atmospheric CH4 are wetlands and irrigated rice fields (1, 2, 32, 40), whose flooded soils provide the conditions required for the activity and growth of methanogenic archaea. Once formed, CH4 diffuses along concentration gradients to the surface and is eventually released into the atmosphere. However, emission may be significantly reduced by the activity of CH4-oxidizing bacteria. In some marine and inland saline waters, anaerobic CH4 oxidation takes place (22, 23), but in the vast majority of freshwater and terrestrial ecosystems, CH4 oxidation is exclusively an aerobic process. Hence, net emission is the result of two counteracting processes: emission 5 production 2 oxidation. One microenvironment where CH4-oxidizing bacteria occur is the millimeter-thick oxic surface layer of flooded soils and sediments. Oxidation in this layer may be very effective, consuming between 70 and 90% of the CH4 that is produced in the anoxic soil layers (4, 10, 18, 28, 30, 42). The rhizosphere of wetland plants is another environment where both O2 and CH4 occur, and plant-associated CH4 oxidation has been found in a variety of wetland plants, including rice (13, 18, 20, 27, 28). CH4 oxidation in the rhizosphere may consume more than 90% of CH4 produced, but typically values of around 30% are reported (3, 13, 15, 19, 20, 28, 50).
While most authors agree that CH4 oxidation significantly reduces CH4 emissions (9, 35, 40), the quantification of CH4 oxidation has proved to be rather difficult. One method is to measure rates of CH4 production in soil slurries and to compare these to the rates of CH4 emission (20, 45). However, disrupting and slurrying a soil may bias the rates in different ways. Other methods attempt to inhibit CH4 oxidation. The difference between the emission rates before and after application of an inhibitor is taken as the oxidation rate. Usual techniques include the elimination of O2 (18, 19) or the application of C2H2 (50) as a suicide inhibitor of the CH4 monooxygenase (39). There is an ongoing discussion about the possible bias of these methods (13, 15). While the former method may stimulate methanogenesis, the latter may instead reduce methanogenesis, because C2H2 inhibits methanogenic archaea also (48). In the search for a useful inhibitor of CH4 oxidation with which to investigate the significance of CH4-oxidizing bacteria, methyl fluoride (fluoromethane [CH3F]) was introduced as a potential selective inhibitor (35, 36) and has been used to assess the importance of CH4 oxidation (3, 11, 13, 15, 17, 30, 44). In experiments with methanogenic rice paddy soil, it was found that CH3F did indeed inhibit CH4 oxidation but simultaneously also inhibited CH4 production (17). CH3F has similarly been reported to inhibit CH4 production by slurries of a marine sediment (36) and by samples from a peatland (28). Indeed, CH3F has been discussed as an inhibitor that may be selective against aceticlastic methanogens (17). We investigated the effect of CH3F on pure and mixed cultures of anaerobic microorganisms, including methanogenic archaea, to confirm these observations. MATERIALS AND METHODS Medium preparation. The anoxic, bicarbonate-buffered, sulfide-reduced mineral medium FM, containing vitamins and trace elements, was prepared as described previously (25). Trace element solution SL10 (52) was usually used, but trace element solution SL9 (49) was substituted as noted below. For some media, Na2S was omitted and the medium was reduced with 0.25 mM titani-
* Corresponding author. Present address: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia. Phone: 61 (3) 9344 5706. Fax: 61 (3) 9347 1540. E-mail:
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TABLE 1. Growth inhibition of various microorganisms by 1 kPa of CH3Fa Organism or syntrophic association
Energy source c,d
Product measured
Inhibitionb
Acetate Acetate Acetate Hydrogene Acetated Hydrogend,e Acetate
CH4 CH4 CH4 CH4 CH4 CH4 CH4
1 1 1 2 1 2 1
Desulfovibrio vulgaris Marburg Desulfotomaculum orientis Singapore 1 Desulfotomaculum sp. strain VeAc5 Pelobacter acetylenicus WoAcy1
Hydrogene Formatee Hydrogene Hydrogene Formatee Hydrogene Hydrogene Formatee Hydrogen Lactate Hydrogen Hydrogen Ethanol Hydrogen 1 sulfatee Hydrogen 1 sulfatec, f Acetate 1 sulfate Acetoin
CH4 CH4 CH4 CH4 CH4 CH4 CH4 CH4 Acetate Acetate Acetate Acetate Acetate Sulfide Sulfide Sulfide Acetate
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
Pelobacter acetylenicus WoAcy1 1 Methanobacterium formicicum MF Unidentified bacterium WoAct 1 Methanosaeta concilii GP6
Ethanol Acetonec
CH4 CH4
2 1
Methanosaeta concilii GP6 Methanosaeta sp. strain VeAc9 Methanosarcina barkeri MS Methanosarcina mazei S-6 Methanosarcina sp. strain VeA23 Methanospirillum hungatei JF1 SK Methanobacterium formicicum MF Methanobacterium bryantii MoH Methanobacterium sp. strain VeH52 Acetobacterium woodii WB1 Acetobacterium carbinolicum WoProp1 Sporomusa ovata H1
a All cultures were grown in sulfide-reduced FM with trace element solution SL10 at 25°C with shaking at 50 rpm in the dark, unless otherwise noted. Cultures were incubated for 500 to 1,000 h. b 1, inhibition of product formation (rate of product formation in the presence of CH3F ,30% of that in cultures without CH3F); 2, no inhibition of product formation (rate of product formation in the presence of CH3F .90% of that in cultures without CH3F). c Trace element solution SL9 used. d Static incubation at 30°C. e Acetate (5 mM) added as a supplementary carbon source. f Titanium(III)-nitrilotriacetic acid used as a reducing agent.
um(III)-nitrilotriacetic acid (33) just prior to inoculation. The pH was adjusted to 7.2 with sterile 1 M HCl or 0.5 M Na2CO3, and the medium was dispensed aseptically into sterile serum bottles. The culture vessels contained 50 ml of medium and a headspace of 70 ml of N2 plus CO2 (80:20, vol/vol) and were closed with butyl-rubber stoppers. Substrate and other supplement stock solutions (1 or 2 M) were sterilized by autoclaving, except for acetoin, which was prepared as a 1 M filter-sterilized (0.2-mm pore size) stock solution. Growth substrates were added just prior to inoculation. Hydrogen was added to the headspace at 60-kPa overpressure. Sodium acetate was used at 50 mM for aceticlastic methanogens and at 20 mM for Desulfotomaculum sp. strain VeAc5 as an energy source. In some cases, 5 mM acetate was added as a supplementary carbon source, as noted. Sodium formate, acetoin, and sodium sulfate were added at 20 mM, and ethanol, sodium L-lactate, and acetone were used at 10 mM (final concentrations). Incubations. Experiments were begun with an inoculum of 5 or 10 ml of a freshly grown, stationary-phase culture. The cultures were incubated with the stoppers downward on a platform rotating horizontally at 50 rpm in the dark at 25°C, unless noted otherwise. The growth conditions and media listed in Table 1 were used in all experiments. CH3F (99%; ABCR, Karlsruhe, Germany) was added to the required concentration with Pressure-Lok series A-2 syringes (Precision Sampling Corp., Baton Rouge, La.). Experiments were carried out at least in duplicate, but for clarity, only one typical set of data is shown in Fig. 1 to 3 and 5. To test the recovery after inhibition by CH3F, the headspace of a culture of Methanosarcina mazei which had been incubated with 1 kPa of CH3F for 734 h was flushed with N2 plus CO2 (80:20, vol/vol) three times, each time for 5 min and each time followed by shaking by hand for 1 min. The residual CH3F partial pressure in the headspace after this procedure was 0.7 Pa. A parallel culture which had been incubated without CH3F was treated similarly but diluted with growth medium to the same optical density as the treated culture, and the volume was adjusted to that of the CH3F-treated culture. This was necessary because the untreated culture had grown, whereas the CH3F-treated culture had not. Both cultures were then incubated without CH3F as normal. Archaeal and bacterial strains. Methanosaeta concilii GP6T (DSM 3671), Methanosarcina barkeri MST (DSM 800), M. mazei S-6T (DSM 2053), Methano-
spirillum hungatei JF1T (DSM 864), Methanobacterium formicicum MFT (DSM 1535), Methanobacterium bryantii MoHT (DSM 863), Pelobacter acetylenicus WoAcy1T (DSM 3246), Acetobacterium woodii WB1T (DSM 1030), Acetobacterium carbinolicum WoProp1T (DSM 2925), Sporomusa ovata H1T (DSM 2662), and Desulfotomaculum orientis Singapore 1T (DSM 765) were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Methanosaeta sp. strain VeAc9, Methanosarcina sp. strain VeA23, Methanobacterium sp. strain VeH52, and Desulfotomaculum sp. strain VeAc5 were isolated from Italian rice paddy soil and identified by comparative sequence analysis of their 16S rRNA genes (24). Methanospirillum hungatei SK (DSM 3595), the mixed culture WoAct (38), and Desulfovibrio vulgaris Marburg (DSM 2119) were obtained from Bernhard Schink (Konstanz, Germany). Analytical methods. CH4 and CH3F were quantified by gas chromatography (17). Samples (0.2 ml) were periodically withdrawn with Pressure-Lok series A-2 gas sampling syringes and injected immediately into the gas chromatograph. At each sampling time, duplicate samples from each culture were analyzed (difference, ,5%), and the means were calculated. Acetate production was measured by high-pressure liquid chromatography (29). Sulfide production was determined colorimetrically (8). The Bunsen coefficient for CH3F was determined experimentally (11, 17) over a temperature range of 22.5 to 50°C, and, together with other data (5), fitted to the Clarke-Glew-Weiss equation (7, 51) to calculate dissolved CH3F concentrations at 25°C (a 5 0.91) and 30°C (a 5 0.72). Estimation of growth and CH4 production rates. To estimate the growth rate of methanogenic archaea from the CH4 production kinetics, the measured CH4 concentrations were corrected to take into account the inoculum size used: pcCH4 5 (Vi/Vm 3 piCH4) 2 psCH4 1 pmCH4, where pcCH4, piCH4, psCH4, and pmCH4 are the partial pressures of CH4 after correction, in the culture used as the inoculum, at the start of the experiment, and measured, respectively, and Vi and Vm are the liquid volumes of the inoculum used to initiate the experiment and the final medium volume in the experiment, respectively. Growth rates and CH4 production rates were estimated by linear regression of the log of pcCH4 against time, and the standard deviation of the regression coefficient was calculated by using a computer graphics package (Origin version
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FIG. 1. Effect of 1 kPa of CH3F, added to the headspace, on the kinetics of product formation by pure and mixed cultures of anaerobic microorganisms. (a) Methanosaeta concilii GP6 on acetate; (b) Methanosaeta sp. strain VeAc9 on acetate; (c) M. barkerii MS on acetate; (d) unidentified bacterium WoAct plus Methanosaeta concilii GP6 on acetone; (e) Methanospirillum hungatei JF1 on hydrogen plus carbon dioxide, with acetate as a supplementary carbon source; (f) A. woodii WB1 on hydrogen plus carbon dioxide; (g) D. vulgaris Marburg on hydrogen plus sulfate, with acetate as a supplementary carbon source; and (h) P. acetylenicus WoAcy1 plus Methanobacterium formicicum MF on ethanol. Symbols: F, results with 1 kPa of CH3F; E, no CH3F added. For clarity, only one data set is shown for each culture and treatment.
2.94; MicroCal Software, Northampton, Mass.). The cell dry mass at the start of short-term incubation experiments was estimated from the amount of inoculum added and a growth yield of 1.15 g (dry mass) of cells per mol of CH4 produced by the inoculum culture (37).
RESULTS The effect of the addition of 1 kPa of CH3F to the headspace of liquid cultures of various anaerobic bacteria was evaluated by monitoring the production of metabolic end products (Table 1). In all cases, where there was inhibition, the inhibitory effect of CH3F could be clearly seen in the kinetics of end product formation compared to that of control experiments in which no CH3F was added (Fig. 1). The degree of inhibition was not quantified in these experiments and was not the same with all organisms that were inhibited (Fig. 1a to d). Methanogens growing on acetate were clearly inhibited by 1 kPa of CH3F (Table 1 and Fig. 1a to c), which corresponds to dissolved CH3F concentrations of 365 mM at 25°C and 285 mM at 30°C. Methanogens growing on hydrogen or formate, homoacetogenic bacteria, sulfate-reducing bacteria, and the fermentative bacterium P. acetylenicus WoAcy1 were not inhibited at these CH3F concentrations (Table 1 and Fig. 1e to g). An ethanol-degrading syntrophic culture, consisting of the ethanol-fermenting bacterium P. acetylenicus WoAcy1 and the hydrogen- and formate-utilizing methanogen Methanobacterium formicicum MF (46), was not inhibited, but the mixed culture WoAct, which consists of an unidentified acetone-fermenting bacterium and the aceticlastic methanogen Methano-
saeta concilii GP6 (38), was inhibited by 1 kPa of CH3F (Table 1 and Fig. 1d and h). M. barkeri MS growing on hydrogen in the presence of acetate degraded the acetate to a concentration of about 850 mM in the absence of CH3F, about the threshold concentration expected for Methanosarcina spp. (26). When CH3F was present, CH4 was still formed, but acetate utilization was minimal (Fig. 2), suggesting that only hydrogenotrophic methanogenesis was occurring. The addition of 1 kPa of CH3F inhibited CH4 production from acetate by M. mazei S-6. When the CH3F was removed from the culture after 734 h by flushing the culture vessel headspace with N2 plus CO2 (80:20, vol/vol), CH4 production commenced with the same kinetics as in a similarly treated control which had not been inhibited with CH3F (Fig. 3). We tested the effects of different CH3F partial pressures on the growth rates, as measured by CH4 production, of three methanogens (Fig. 4). Methanosaeta concilii GP6 growing on acetate was most sensitive to inhibition by CH3F, with a 50% reduction of the growth rate observed at about 0.6 kPa of CH3F. Methanospirillum hungatei JF1 growing on hydrogen was much less sensitive to CH3F, with a 50% inhibition of the growth rate occurring at about 8.7 kPa of CH3F. M. mazei S-6 growing on acetate was more sensitive to CH3F (50% inhibition at 2.3 kPa of CH3F) than when growing on hydrogen (15.6 kPa of CH3F resulting in only a 37% inhibition). To differentiate between an inhibition of growth only and an inhibition of methanogenesis, the specific rate of CH4 produc-
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FIG. 2. Effect of 1 kPa of CH3F (closed symbols) on CH4 production (F and E) and acetate utilization (å and Ç) by M. barkeri MS growing on acetate plus hydrogen. In the control experiments (open symbols), incubation was done without CH3F addition. For clarity, only one data set is shown for each treatment.
tion was measured over the initial 160 h in freshly inoculated cultures of Methanosaeta concilii GP6. The doubling time in the logarithmic phase of growth under the incubation conditions used was estimated at 170 to 180 h. The specific rate of CH4 production from acetate in treatments with 1 kPa of CH3F was 0.86 mmol of CH4 per mg (dry mass) of cells per h, while in parallel treatments without CH3F the specific rate of CH4 production from acetate was 7.33 mmol of CH4 per mg (dry mass) of cells per h (Fig. 5). DISCUSSION CH3F at a partial pressure of about 1 kPa was an inhibitor of growth of and CH4 production by methanogenic archaea growing with acetate as their sole energy source. At this concentration, CH4 formation from hydrogen or from formate by pure cultures was not inhibited. M. barkeri, a methanogen which can grow on either hydrogen or acetate, was inhibited only when growing with acetate. When growing mixotrophically, methanogenesis from acetate appeared to be specifically inhibited, without inhibition of CH4 formation from hydrogen plus carbon dioxide. A mixed culture growing on ethanol, in which P. acetylenicus degraded ethanol to acetate plus hydrogen and Methanobacterium formicicum utilized the hydrogen produced, was not inhibited by CH3F, as evidenced by continued CH4 production. Ethanol degradation in such cultures proceeds only when the hydrogen partial pressure is kept below 3.2 kPa (46). This shows that the lack of inhibition of hydrogenotrophic methanogenesis by CH3F at 1 kPa was not due to the high hydrogen partial pressure (60 kPa) used in batch culture experiments. In contrast, the acetone-fermenting mixed culture WoAct, consisting of an unidentified bacterium which degrades acetone to acetate and the obligately aceticlastic methanogen Methanosaeta concilii (38), was inhibited by 1 kPa of CH3F. Steady-state acetate concentrations in growing cultures of this acetate syntrophy are less than 100 mM (38). Long-term incubations showed that the growth of aceticlastic methanogenic archaea was inhibited by CH3F. Shorter incubations showed that CH4 production from acetate was also inhibited in the absence of growth. This suggests that CH3F
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directly affects some step(s) in the pathway of methanogenesis. Although acetate utilization by methanogenic archaea was inhibited, acetate utilization by the sulfate-reducing bacterium Desulfotomaculum sp. strain VeAc5 was not. Desulfotomaculum spp. degrade acetate via the acetyl coenzyme A-carbon monoxide dehydrogenase pathway (43, 47), a pathway which has many biochemical similarities to the pathway of methanogenesis from acetate (16). Acetate formation by homoacetogenic bacteria, which also has biochemical similarities to the pathway of methanogenesis from acetate (14), was also not inhibited by 1 kPa of CH3F. The inhibition of CH4 production by CH3F appears not to result in irreversible inhibition of the methanogens, since removal of the CH3F even after more than 700 h resulted in the recovery of CH4 production. A similar reversible inhibition was also found in methanogenic slurries of Italian rice paddy soil treated with CH3F (17). Different methanogens appear to be inhibited to differing degrees by various amounts of CH3F. However, with about 1 kPa of CH3F, used to date in the investigation of CH4 fluxes from natural systems (17, 36, 44), aceticlastic methanogens are inhibited while hydrogenotrophic methanogens are not. Methanolobus taylorii (34) growing on trimethylamine was not inhibited at similar CH3F concentrations but was inhibited at higher concentrations (36). The selective inhibition of aceticlastic methanogenesis at low CH3F concentrations in our study agrees with earlier findings that acetate failed to stimulate methanogenesis in slurries of Italian rice paddy soil incubated with CH3F (17). Acetate accumulated in rice paddy soil slurries incubated with CH3F, suggesting that its degradation was inhibited to a greater extent than its production. In this anoxic rice paddy soil, about 80% of CH4 is produced from acetate (6, 42). Since the amount of CH4 formed in the untreated controls balanced the amount of acetate which accumulated in the inhibited slurries (17), and since 1 mol of CH4 is formed from 1 mol of acetate, it is apparent that the effect of CH3F in soil slurries is to inhibit more or less completely aceticlastic methanogenesis. These results are translatable to other observations. The inhibition of CH4 formation by washed rice (Oryza sativa) roots incubated with CH3F (17) strongly suggests an important role
FIG. 3. Reversibility of inhibition by CH3F of CH4 production from acetate by M. mazei S-6. After incubation with 1 kPa of CH3F (F) for 734 h, CH3F was removed (see Materials and Methods). Controls (E) were not incubated with CH3F. For clarity, only one data set is shown for each treatment.
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CH3F, no 14CH4 was formed from [2-14C]acetate (36). The findings of both this study and a previous study (17) show that great care must be taken when CH3F is used as an inhibitor of CH4 oxidation in undefined systems, since it can also lead to an inhibition of CH4 production. When CH3F is used to investigate the components of CH4 emission, this could then result in an underestimation of the importance of CH4 oxidation, especially where acetate is the major substrate for methanogenesis. Our findings also suggest a possible use of CH3F as a selective inhibitor of aceticlastic methanogenesis by careful choice of the concentrations used, especially for the rapid determination of the role of acetate as a CH4 precursor. Major points to note are that the degree of inhibition of aceticlastic methanogenesis is variable (depending on the strain tested) and that at higher partial pressures of CH3F, hydrogenotrophic methanogenesis is also inhibited. REFERENCES
FIG. 4. Effects of different CH3F concentrations on the growth rate, estimated from the CH4 production rate (see Materials and Methods), of Methanospirillum hungatei JF1 growing on hydrogen plus carbon dioxide (Ç), Methanosaeta concilii GP6 growing on acetate (å), and M. mazei S-6 growing on hydrogen plus carbon dioxide (F) and on acetate (E). The error bars (standard deviations of the regression coefficients) are not shown if they are smaller than the symbol.
for aceticlastic methanogens, in contrast to the root-associated methanogenesis of cottontail (Typha latifolia), which was not inhibited by CH3F (17). The addition of CH3F to pieces of algal mat from the hypersaline Solar Lake did not result in the inhibition of CH4 formation (17), which suggests that aceticlastic methanogenesis is insignificant in this habitat. This agrees well with the finding that acetate failed to stimulate methanogenesis by pieces of the same algal mat, in contrast to trimethylamine, which greatly stimulated CH4 production (41). CH3F (at 1.7 kPa) has been reported not to inhibit methanogenesis from trimethylamine (36). These findings extend, verify, and explain an earlier report of the inhibition of CH4 production by some methanogenic systems (17) and are in agreement with the report that under
FIG. 5. Inhibition of CH4 production by Methanosaeta concilii GP6 in a short-term incubation measuring CH4 production by 1 mg (dry mass) of cells in 50 ml of medium, with 1 kPa of CH3F (F) or with no added CH3F (E).
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