Bioenergetics of Methanogenesis from Acetate by Methanosarcina Barkerit

Home , Sodium acetate

JOURNAL OF BACTERIOLOGY, Mar. 1988, p. 1369-1372 Vol. 170, No. 3 0021-9193/88/031369-04$02.00/0 Copyright © 1988, American Society for Microbiology Bioenergetics of Methanogenesis from Acetate by Methanosarcina barkerit SUSANNE PEINEMANN, VOLKER MULLER, MICHAEL BLAUT, AND GERHARD GOTTSCHALK* Institut fur Mikrobiologie der Universitat Gottingen, Grisebachstrasse 8, D-3400 Gottingen, Federal Republic of Germany Received 24 June 1987/Accepted 17 November 1987 Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient, however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+o05/Na+1n) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.

Recently, it was shown that methanogenesis from H2 plus anaerobic techniques of Hungate (9) as described recently methanol, H2 plus trimethylamine, and H2 plus formalde- (19). The gas atmosphere was N2-C02 (80:20, vol/vol). hyde by resting cells of Methanosarcina barkeri is coupled Growing on sodium acetate, M. barkeri had a doubling time to ATP formation by a chemiosmotic mechanism (2, 3, 19). of 24 h and the growth yield was 2.5 mg (cell dry This conclusion was based on the following findings. (i) The weight)/mmol of methane formed. addition of the protonophore tetrachlorosalicylanilide (TCS) Cell suspensions of M. barkeri in 100 mM sodium pipera- to resting cells ofM. barkeri forming methane from methanol zine-N,N'-bis(2-ethanesulfonic acid) (sodium PIPES) buffer, plus H2, formaldehyde plus H2, or trimethylamine plus H2 pH 6.7, containing 2 ml of titanium(III) citrate solution were caused a dissipation of the proton motive force (Ap) and a prepared as described previously (19). The protein concen- decrease in the intracellular ATP content, whereas mnethane tration of the final cell suspension was 13 to 14 mg/ml. For formation was stimulated. (ii) The ATPase inhibitor N,N'- experiments in which the sodium dependence was tested, dicyclohexylcarbodiimide (DCCD) inhibited methane forma- cell suspensions were prepared in 20 mM imidazole buffer, tion and ATP synthesis but left Ap intact. (iii) Addition of pH 6.8, supplemented with 25 mM KCI. The protein content TCS to cell suspensions incubated with DCCD restored their was determined by the method of Schmidt et al. (22). ability to form methane. We have extended these studies Experimental conditions. Effects of DCCD and TCS were now to cells of M. barkeri grown on acetate. Important studied as described previously (19). To study the effect of differences have been encountered in comparison to the sodium ions, 20 mM potassium acetate served as the sub- substrate combination methanol plus H2; they are reported strate and NaCl was added as indicated with a syringe from in this publication. a 3 M stock solution. H2 formation from CO plus H20 by cell It has been known for some time now that the growth of suspensions was measured in the presence of 20 mM potas- methanogenic bacteria and methane formation depend on sium 2-bromoethanesulfonate (BES)-1 mM propyl iodide-20 sodium ions (20); an involvement of Na+ in the process of mM KCI in the absence of light (5). The reaction was started ATP synthesis was envisaged. However, we have shown by the addition of 5 ml of gaseous CO. A potassium BES that ATP synthesis during methanogenesis from methanol solution was prepared from the sodium salt by ion-exchange plus H2 does not depend on the presence of Na+ (4). chromatography on Dowex 50 WX8. Fifty milliliters of 0.2 M Recently, one site ofNa+ involvement was identified. Na+ is sodium BES was mixed with 100 ml ofDowex 50 WX8, 20/50 required for methyl group oxidation during dismutation of mesh, and incubated with gentle stirring for 15 min. The BES methanol to methane and carbon dioxide (4). It was of solution was subsequently separated from the resin by interest to study the effect of Na+ on methanogenesis from filtration. The resulting solution was passed twice through a acetate. 5-ml column filled with Dowex 50 WX8, 100/200 mesh. Finally, the pH of the BES solution was adjusted with KOH MATERIALS AND METHODS to 7.0. The sodium concentration in this solution was below M. barkeri Fusaro (DSM 804) was grown on 100 mM 1 mM. sodium acetate as the sole carbon and energy source. The Methane, acetate, and H2 concentrations were determined medium described by Hippe et al. (8) was prepared by the by gas chromatography (4, 17), and ATP concentration was determined by the luciferin luciferase assay as described * Corresponding author. previously (13, 19). t This paper is dedicated to H. A. Barker (University of Califor- A* and ApH were estimated from the transmembrane nia, Berkeley) on the occasion of his 80th birthday in recognition of equilibrium distribution of the lipophilic cation [14C]tet- his pioneering studies on methanogenic bacteria. raphenylphosphonium bromide and the weak acid [14C]- 1369 1370 PEINEMANN ET AL. J.- BACTERIOL.

Republic of Germany. TCS was from Eastman Kodak Co., Rochester, N.Y. [7-'4C]benzoic acid, ["4C]tetraphenylphos- phonium bromide, 3H20, ['4C]sucrose, and 22Na' were purchased from New England Nuclear Corp., Dreieich, Federal Republic of Germany. The silicon oil was from Roth, Karlsruhe, Federal Republic of Germany. Gases were 2 ~~~~~~~~~~~~~15 from Messer-Griesheim, Kassel, Federal Republic of Ger- many. 50- 10 RESULTS Generation of a proton motive force during methanogenesis from acetate and effect of DCCD. The following experiments 0 -0 were done with M. barkeri Fusaro that had been transferred +20- in media with acetate as the sole carbon and energy source for more than 6 months. Cell suspensions of this strain 0 100 200 300 formed methane from acetate under N2 at a maximal rate of Time (min) 90 to 120 nmol/min per mg of protein. After a lag phase, this FIG. 1. Magnitude and composition of Ap during methane for- rate was constant for approximately 100 min. Methane mation from acetate by cell suspensions of M. barkeri. A 10-ml cell in suspension (1.4 mg of protein per ml) was made in 100 mM sodium formation was coupled to an increase the intracellular PIPES buffer, pH 6.7; 15 mM acetate was added at the time ATP concentration to a level of 3.5 to 4.0 nmol/mg of indicated by the arrow. Symbols: , -62 ApH; *, /v+; A, Ap; *, protein. In the absence of acetate, the cells had an ATP CH4. content of only 0.8 to 0.9 nmol/mg of protein. H2 at concentrations of 0.5% in the gas phase completely blocked methanogenesis from acetate (data not shown). benzoic acid, respectively, by the method of Rottenberg (21) The proton motive force (Ap) increased to a steady-state as described previously (19). level of -120 mV upon the addition of acetate. After Determination of the extracellular and intracellular sodium termination of methane formation, this steady-state level did concentrations. A portion of the cell suspension was freed of not decline rapidly but stayed at a high level for more than 3 cells by centrifugation. The sodium concentration was mea- h (Fig. 1). Ap consisted predominantly of the membrane sured with a sodium electrode (Orion Research AG, Kus- potential (At); the transmembrane gradient of H+ (ApH) was nacht, Switzerland) connected to an ion meter (Orion Re- small and in the opposite orientation. search AG). The intracellular Na+ concentration was The addition of the uncoupler TCS to cell suspensions determined by using a membrane filtration method. Cells in which were actively forming methane from acetate resulted the mid-log phase were harvested as described above and in an inhibition of methane formation and in a rapid decrease suspended in 20 mM imidazole hydrochloride buffer, pH 6.5, in Ap (Fig. 2), as well as a rapid decrease in the intracellular containing 20 mM MgCl2 and 25 mM NaCl. Experiments ATP concentration (data not shown). DCCD, an inhibitor of were performed anaerobically under N2 at 37°C in 18-ml proton-translocating ATPases of procaryotic organisms, in- serum tubes with 1 ml of the buffer described above. A cluding M. barkeri (10), also affected methanogenesis from 100-,ul portion ofthe concentrated cell suspension was added acetate. The formation of methane stopped, and, in contrast anaerobically. After preincubation for 15 min on a rotary to our expectation, Ap did not remain constant but decreased shaker, 10 RI of potassium acetate (2.5 M) and 10 ,ll of gradually (Fig. 2). This observation was different from carrier-free 22NaCl (1 uCi/Wpl) were added and the cells were allowed to metabolize for 60 min. Subsequently, 100-,ul aliquots were withdrawn with a syringe. To determine CO-dependent efflux of 22Na', cells were incubated in the buffer described above containing 10 ,uCi of -120 22Na' for 18 h on ice. Subsequently, the reaction mixture was kept in darkness in a water bath at 37°C, and 20 mM potassium BES and 1 mM propyl iodide were added. The _9 reaction was started by the addition of 1.6 ml of gaseous CO. 2 Acetate-dependent efflux of 22Na+ was measured as follows. - Cells were harvested and suspended in 3 ml of 20 mM 6 imidazole buffer, pH 6.5, containing 20 mM MgCl2, 250 mM NaCl, and 5 mM dithioerythritol. A 100-,lI portion ofthis cell suspension was incubated in the presence of 10 ,Ci of 22Na+ in 2.3-ml serum tubes under N2 for 24 h on ice. At 20 min prior to zero time, the suspension was brought to 37°C and was diluted, at zero time, by the addition of 1 ml of 20 mM 100 150 imidazole buffer, pH 6.5, containing 20 mM MgCl2, 250 mM Time (min) sucrose, and 5 mM dithioerythritol. Potassium acetate was FIG. 2. Effect of TCS and DCCD on methane formation from acetate and on Ap. Experimental conditions were as described in the added to a final concentration of 50 mM. Determination of legend to Fig. 1. At the time indicated by the closed arrow, 15 mM the radioactivity and preparation of the membrane filters acetate was added. TCS (final concentration, 10 ,IM) or DCCD (25 were done as described previously (18). nmol/mg of protein) was added at the time indicated by the open Chemicals and gases. PIPES, DCCD, luciferin luciferase, arrow. Closed symbols, TCS experiment; open symbols, DCCD and BES were purchased from Sigma, Taufkirchen, Federal experiment; 0 and 0, CH4; A and A, Ap. VOL. 170, 1988 METHANOGENESIS FROM ACETATE 1371

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0 20 40 60 a0 100 120 0 10 20 30 40 Time (min) Time (min) FIG. 4. Effect of Na+ on intracellular ATP concentration and on (A) methane formation from acetate or (B) H2 formation from CO. At the times indicated by solid arrows, 20 mM potassium acetate (A) Tine(min) or 5 ml of gaseous CO (B) was added to 10-ml cell suspensions. Symbols: 0, CH4, and U, ATP at 0.7 mM Na+; 0, CH4, and 0, ATP FIG. 3. Effect of DCCD on methanogenesis and on Ap of M. at 21 mM Na+; open arrow, Na+ in one cell suspension which was barkeri incubated with acetate and methanol under an H2 atmo- increased from 0.7 to 25 mM. Protein concentrations: (A) 1.1 mg/ml; sphere. Experimental conditions were as described in the legends to (B) 0.7 mg/ml. Fig. 1 and 2. Open arrow, addition of DCCD; open and closed symbols, presence and absence, respectively, of DCCD; 0 and 0, CH4; A and A, Ap. intracellular Na+ concentration did not change. Correspond- ingly, cells incubated in the presence of 25 mM 22NaCl extruded Na+ uphill against a concentration gradient upon observations made with M. barkeri forming methane from the addition of CO (Fig. SB). In a number of experiments, H2 plus methanol (2) or from H2 plus trimethylamine (19). the magnitude of the Na+ gradient was determined during It was possible that the decline in Ap in the presence of methanogenesis from acetate; it amounted to 4.0 + 0.4 mM DCCD was due to an uncoupling effect of acetate. There- (Na'out/Na%) at an external Na+ concentration of 25 mM. fore, we examined whether acetate had an effect on Ap in This transmembrane Na+ gradient was abolished by the acetate-grown cells which were energized with methanol addition of 10 ,uM TCS (data not shown). plus H2 and subsequently treated with DCCD (Fig. 3). A decline in Ap was not observed under these conditions. DISCUSSION Role of Na+ in methanogenesis from acetate. Possible reasons for the observed Na+ dependence of methane for- In some anaerobic environments, acetate is the most mation from acetate were investigated. M. barkeri cells were important methanogenic substrate (6). However, the path- suspended in buffer containing various amounts of NaCl, 20 way of acetate conversion into methane and carbon dioxide mM potassium BES, and 1 mM propyl iodide. The addition is still not known in detail, and the bioenergetics of this of gaseous CO to such suspensions led to H2 formation conversion have not been studied until now. irrespective of the external Na+ concentration (data not Here, we showed that methanogenesis from acetate is shown). This finding indicated that the CO dehydrogenase accompanied by the generation of a proton motive force and was functional in the absence of Na+ but did not exclude the an increase in the intracellular ATP level. The inhibitor possibility that at low external Na+ concentrations an un- studies with DCCD allowed us to conclude that with acetate coupling occurred between H2 formation and ATP synthesis. as the methanogenic substrate, ATP is synthesized by a Therefore, the effect of Na+ on the intracellular ATP content during methanogenesis from acetate and during H2 formation from CO plus H20 was investigated. In the presence of 0.7 mM NaCl, little methane was formed from acetate and the intracellular ATP level was low compared with that in the presence of 21 mM NaCl. Addition of NA+ to a final concentration of 25 mM to each suspension led to methane formation, as well as to net ATP synthesis (Fig. 4A). On the E other hand, the same rate of ATP synthesis and the same -A: intracellular ATP concentration were achieved when CO served as the energy source (Fig. 4B). The intracellular sodium concentration during this fermen- tation was determined. Cells of M. barkeri were allowed to equilibrate in a buffer containing 250 mM 22NaCl for 24 h on 0 10 20 30 40 50 60 0 10 20 30 40 ice. During this period, the cells took up Na+ to a final Time (min) TimeImin) concentration of 78 mM. At zero time, the suspension was FIG. 5. Sodium extrusion in M. barkeri as a result of (A) diluted 10-fold by the addition of NaCl-free buffer, thus methanogenesis from acetate or (B) H2 formation from CO. For creating an outwardly directed Na+ gradient. The addition of experimental details, see Materials and Methods. Protein content of acetate resulted in a decrease in the intracellular Na+ final cell suspensions: (A) 1.9 mg/ml; (B) 1.7 mg/ml. Symbols: *, concentration (Fig. SA). In the absence of a substrate, the Na+in in the presence of acetate or CO; *, Na+in in their absence. 1372 PEINEMANN ET AL. J. BACTERIOL.

chemiosmotic mechanism. Of note is the gradual decrease in 2. Blaut, M., and G. Gottschalk. 1984. Coupling of ATP synthesis Ap, which was not observed with the substrate combination and methane formation from methanol and molecular hydrogen methanol plus H2 (2) but was observed with the combination in Methanosarcina barkeri. Eur. J. Biochem. 141:217-222. CO2 plus H2 (3). In the case of methanogenesis from acetate, 3. Blaut, M., and G. Gottschalk. 1984. Proton-motive force-driven synthesis of ATP during methane formation from molecular it may be explained as follows. After depletion of the ATP hydrogen and formaldehyde or carbon dioxide in Methanosar- pool, acetate can no longer be activated. If Ap decreases as cina barkeri. FEMS Microbiol. Lett. 24:103-107. the result of transport or leakage processes, it cannot be 4. Blaut, M., V. Muller, K. Fiebig, and G. Gottschalk. 1985. replenished by methanogenesis because activated acetate, Sodium ions and an energized membrane are required by presumably acetyl coenzyme A, is not available. The de- Methanosarcina barkeri for the oxidation of methanol to the crease in Ap is not due to an uncoupling effect of acetate, level of formaldehyde. J. Bacteriol. 164:95-101. since it did not occur with acetate plus methanol as the 5. Bott, M., B. Eikmanns, and R. K. Thauer. 1986. Coupling of substrate. carbon monoxide oxidation to CO2 and H2 with the phosphor- The addition of the protonophore TCS resulted in an ylation of ADP in acetate-grown Methanosarcina barkeri. Eur. J. Biochem. 159:393-398. inhibition of methanogenesis and a decrease in both Ap and 6. Cappenberg, T., and H. Prins. 1974. Interrelationships between the intracellular ATP content. This is in contrast to metha- sulfate reducing and methane-producing bacteria in bottom nogenesis with the substrate combination methanol plus H2 deposits of a freshwater lake. III. Experiments with 14C-labeled (2) but is similar to that with methanol (4) and to that with H2 substrates. Antonie van Leeuwenhoek J. Microbiol. Serol. plus C02 (3). This inhibition is conceivable if acetate must be 40:457-459. activated prior to its conversion to CO2 and methane. 7. Eikmanns, B., and R. K. Thauer. 1984. Catalysis of an isotopic Not yet understandable is the requirement of methanoge- exchange between CO2 and the carboxyl group of acetate by nesis from acetate for a Na+ gradient. The Na+ gradient in Methanosarcina barkeri grown on acetate. Arch. Microbiol. M. barkeri has been shown not to be involved in pH 138:365-370. regulation at acidic pH values or in ATP synthesis (4). Since 8. Hippe, H., D. Caspari, K. Fiebig, and G. Gottschalk. 1979. Utilization of trimethylamine and other N-methyl compounds this gradient is abolished by the proton conductor TCS, it is for growth and methane formation by Methanosarcina barkeri. presumably the result of secondary Na+ translocation driven Proc. Natl. Acad. Sci. USA 76:494-498. by the Na+/H+ antiporter, as has been shown for M. barkeri 9. Hungate, R. E. 1969. A roll tube method for cultivation of strict catabolizing methanol (18). From the pathway leading from anaerobes, p. 117-132. In J. R. Norris and D. W. Ribbons (ed.), acetate to methane and carbon dioxide the following reac- Methods in Microbiology, vol. 3b. Academic Press, Inc., New tions can be identified as functioning without a Na+ gradient: York. the conversion ofacetate to acetyl coenzyme A, as catalyzed 10. Inatomi, K. I. 1986. Characterization and purification of the by the enzymes acetate kinase and phosphotransacetylase membrane-bound ATPase of the archaebacterium Methanosar- (12, 15); the reduction of methyl coenzyme M, which was cina barkeri. J. Bacteriol. 167:837-841. recently in 11. Keltjens, J. T., and C. van der Drift. 1986. Electron transfer identified as an intermediate methanogenesis reactions in methanogens. FEMS Microbiol. Rev. 39:259-303. from acetate (16); ATP synthase (4); and the CO dehydro- 12. Kenealy, W. R., and J. G. Zeikus. 1982. One-carbon metabolism genase (reference 5 and this study). However, at least two in methanogens: evidence for synthesis of a two-carbon cellular reactions remain that could be driven by a sodium motive intermediate and unification of catabolism and anabolism in force: the uptake of acetate and the cleavage of acetyl Methanosarcina barkeri. J. Bacteriol. 151:932-941. coenzyme A. The latter reaction is endergonic, with a AG0 13. Kimmich, G. A., J. Randles, and J. S. Brand. 1975. Assay of of +40.3 kJ per reaction (11). An electrochemical Na+ picomole amounts of ATP, ADP and AMP using the luciferase potential could drive this reaction as it drives the oxidation enzyme system. Anal. Biochem. 69:187-206. of methanol to the level of formaldehyde in M. barkeri (V. 14. Krzycki, J. A., and J. G. Zeikus. 1984. Acetate catabolism by Muller, unpublished results). Such a conclusion is very Methanosarcina barkeri: hydrogen-dependent methane produc- it it tion from acetate by a soluble cell protein fraction. FEMS tempting because readily explains why has been very Microbiol. Lett. 25:27-32. difficult to develop a cell-free system that catalyzes the 15. Laufer, K., B. Eikmanns, U. Frinmer, and R. K. Thauer. 1987. conversion of acetyl coenzyme A to methane and carbon Methanogenesis from acetate by Methanosarcina barkeri: ca- dioxide. Baresi (1) reported that this is possible only with talysis of acetate formation from methyl iodide, CO2 and H2 by pelleted membrane fractions. In another system, H2 was the enzyme system involved. Z. Naturforsch. 42:360-373. required (14) which was shown to inhibit methane formation 16. Lovley, D. R., R. H. White, and J. G. Ferry. 1984. Identification from acetate in M. barkeri Fusaro (7). It is not yet apparent of methyl-coenzyme M as an intermediate in methanogenesis why it is advantageous to M. barkeri (and possibly to other from acetate in Methanosarcina spp. J. Bacteriol. 160:521-525. methanogenic bacteria) to use a proton motive force for ATP 17. Muller, V., M. Blaut, and G. Gottschalk. 1986. Utilization of synthesis and a sodium motive force for certain reactions methanol plus hydrogen by Methanosarcina barkeri for metha- involved in methane formation from methanol, CO2 plus H2, nogenesis and growth. Appl. Environ. Microbiol. 52:269-274. or acetate. 18. Muller, V., M. Blaut, and G. Gottschalk. 1987. Generation of a transmembrane gradient of Na+ in Methanosarcina barkeri. Eur. J. Biochem. 162:461-466. ACKNOWLEDGMENTS 19. Muller, V., G. Kozianowski, M. Blaut, and G. Gottschalk. 1987. We are indebted to the staff of the Zentrales Isotopenlabor der Methanogenesis from trimethylamine + H2 by Methanosarcina Universitat Gottingen for their helpful advice in performing the barkeri is coupled to ATP formation by a chemiosmotic mech- 22Na' experiments. The skillful technical assistance of K. Freisl is anism. Biochim. Biophys. Acta 892:207-212. gratefully acknowledged. We are indebted to G. Kozianowski for 20. Perski, H. J., P. Schonheit, and R. K. Thauer. 1982. Sodium assistance. dependence of methane formation in methanogenic bacteria. This work was supported by a grant from the Deutsche For- FEBS Lett. 143:323-326. schungsgemeinschaft. 21. Rottenberg, H. 1979. The measurement of membrane potential and ApH in cells, organelles, and vesicles. Methods Enzymol. LITERATURE CITED 55:547-569. 1. Baresi, L. 1984. Methanogenic cleavage of acetate by lysates of 22. Schmidt, K., S. Liaanen-Jensen, and H. G. Schlegel. 1963. Die Methanosarcina barkeri. J. Bacteriol. 160:365-370. Carotinoide der Thiorhodaceae. Arch. Microbiol. 46:117-126.