Vol. 132, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, OCt. 1977, P. 118-126 Copyright © 1977 American Society for Microbiology

Carbon Monoxide Oxidation by Methanogenic Bacteria L. DANIELS,' G. FUCHS,lt R. K. THAUER,2* AND J. G. ZEIKUS'* Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706,1 and Fachbereich BiologieMikrobiologie, Philipps-Universitdit, Marburg, Federal Republic of Germany2

Received for publication 5 July 1977

Different species of methanogenic bacteria growing on C02 and H2 were shown to remove CO added to the gas phase. Rates up to 0.2 ,umol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to C02 dnd CH4 according to the following equation: 4CO + 2H20 1CH4 + 3CO2. However, growth was slight, and the growth rate on CO was only 1% of that observed on H2/CO2. Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an acitve factor 420 (F420)dependent hydrogenase (7.7 ,umol/min per mg of protein at 350C) and a COdehydrogenating enzyme (0.2 ,tmol/min per mg of protein at 350C) that catalyzed the reduction of F,20 with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F,,0, viologen dyes were effective electron acceptors for the enzyme. The apparent Km for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (AHt) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 AM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F420 as the electron acceptor. -.

It has been established (9, 10, 33) that methanogenic bacteria can reduce CO with H2 to form methane according to reaction I: CO + 3H2 -- CH4 + H20; AGO' = -36.0 kcal/mol of CO (I) (-150 kJ/mol of CO) (AGO' values were calculated from AlGf vali !S [35] for carbon monoxide, H2, and CH4 in t e gaseous state.) Fischer et al. (9), using impi e growing cultures of methanogens, first si r_ gested that carbon monoxide was not reduced 0 methane directly by reaction I but at least n part by the combination of reactions II and I i.e., by disproportionation of carbon monox e to C02 and CH4 (reaction IV). CO + H20 -* C02 + H2; AGO' - 4.8 kcal/mol of CO (35) [)

C02 + 4H2 --+CH4 + 2H2O; AGO' = -31.3 kcal/mol of CH4 (35) (III) (-131 kJ/mol of CH4) 4CO + 2H20 -- 3CO2 + CH4; AGO' = -50.5 kcal/mol of CH4 (35) (IV) (-211 kJ/mol of CH4)

Kluyver and Schnellen (19) later proved that carbon monoxide reduction to CH4 proceeded via reactions II and III by studying CH4 formation from carbon monoxide with cultures of Methanosarcina barkeri and Methanobacterium formicicum. However, these authors only conducted experiments with cell suspensions and did not study growth on CO. Uffen (38) has recently reported that M. barkeri and M. formicicum were unable to grow on a solid medium with H2-CO gas (80:20 mixture). This investigation was undertaken to determine whether methanogenic bacteria can grow (-20 kJ/mol of CO) on carbon monoxide as sole energy source and t Permanent address: Fachbereich Biologie-Mikrobio gie, Phillips-Universitit, Marburg, Federal Republic f to obtain an understanding of carbon monoxide Germany. metabolism. Evidence is presented that Meth118

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CO OXIDATION BY METHANOGENIC BACTERIA

anobacterium thermoautotrophicum can grow slowly on carbon monoxide as the sole electron donor. The data indicate that the electron carrier factor 420 (F,20) present in methanogens (6) is reduced by carbon monoxide via an F,20-specific carbon monoxide dehydrogenase activity. Reduced F,20 has been shown to be physiologically reoxidized by enzymes involved in CO2 reduction to methane (36, 37). C02 reduction to methane must be coupled to adenosine 5'-triphosphate synthesis as evidenced by growth (26, 45). Thus, the finding that F.0 is reduced via carbon monoxide oxidation explains how M. thermoautotrophicum grows with carbon monoxide as the energy source. MATERIALS AND METHODS Gases. CO (chemically pure), H2/CO2 (80:20%, vol/vol, premixed), N2 (prepurified), H2 (extra dry), and CO2 (bone dry) were purchased from Matheson, Joliet, Ill.; He (industrial and laboratory grade) was from Chemtron Corp., Chicago, Ill.; and 14CO (40 mCi/mmol) and sodium [14C]carbonate (20 mCi/ mmol) were obtained from Amersham/Searle, Arlington Heights, Ill. Fermentor gas mixtures were made with a Matheson gas proportioner. CO mixtures for growth experiments in test tubes were made by aseptic syringe addition to known gas volumes. Gas mixtures for the determination of the apparent Km value for CO were prepared in a gas burette. Organisms. Cultures of M. barkeri, M. formicicum, and Methanobacterium ruminantium strain PS were provided by M. P. Bryant. M. thermoautotrophicum was the neotype strain AH (45), Methanobacterium arbophilicum was the neotype strain DH1 (44), and Methanosarcina strain UBS was isolated fromn Lake Mendota, Madison, Wis. Clostridium pasteurianum (ATCC 6013) was obtained from the American Type Culture Collection. Growth of bacteria. The cultivation methods used were modifications of the methods developed by Hungate (1, 15). M. thermoautotrophicum was grown at 65°C with a gas mixture of 80% H2/20% CO2 and a bicarbonate-buffered mineral salts medium (final pH 7.4) that contained the following constituents, per liter of distilled water: KH2PO4, 0.15 g; Na2HPO4 * 7H2O, 1.05 g; NH4CI, 0.5 g; MgCl2 * 6H2O, 0.1 g; mineral solution, 10 ml (containing the following constituents per liter of distilled water: nitrilotriacetic acid, 1.5 g; FeCl2 * 4H20, 0.3 g; MnCl2-4H20, 0.1 g; CoCl2, 0.1 g; CaCl2 2H20, 0.1 g; ZnCl2, 0.1 g; CuCl2, 0.02 g; H3BO3, 0.01 g; NaMoO4 * 2H20, 0.01 g; resazurin, 0.0002%; NaHCO3, 4.2 g; and Na2S * 9H20, 0.96 g). The medium was dispensed in 10-ml portions into 28-ml tubes gassed with 80% H2/20% CO2 and then autoclaved. For mass culture, a 14-liter fermentor at 65°C with constant agitation and a continuous H2/CO2 (80/ 20%) gas flow of 200 ml/min was used (5% inoculum). Fermentor-grown cells were harvested in the mid- to late-exponential phase of growth by centrifugation at 35,000 x g in a Sorvall RC-5 (DuPont Instruments) equipped with a KSB continuous-flow sys-

119

tem. After centrifugation the culture medium was decanted, the cell pellet was gassed with H2, and the steel tubes were closed under H2 with a rubber bung and frozen at -20°C. Inocula for experiments with growing cultures were anaerobically removed from an exponential-phase culture that had an absorbancy at 660 nm (A6m) of 0.2 to 0.5. M. barkeri, M. formicicum, M. arbophilicum, and Methanosarcina UBS were grown at 30°C on H2/CO2, using the mineral salts medium described above to which 10 ml of a vitamin solution (40) per liter was added. M. ruminantium was grown at 30°C on H2/ C02, using the medium described by Tzeng et al. (37). C. pasteurianum was grown on a glucose-ammonium-mineral salts medium (16) at 35°C. Bacterial growth was determined in 28-ml tubes, using a Spectronic 20 (Bausch & Lomb) blanked against water at 660 nm. For M. thermoautotrophicum a standard curve relating A., to milligrams (dry weight) of cells showed 0.57 mg (dry weight)/ml of culture at an Am60 of 1.0. Preparation of cell-free extracts. Anaerobic conditions were maintained throughout the entire procedure, and all manipulations were performed in the presence of H2 or an inert gas phase at about 40C. Cell-free extracts of M. thermoautotrophicum, M. arbophilicum, and Methanosarcina UBS were prepared by passing anaerobically a cell suspension (1 part [by weight] of frozen cells suspended in 3 parts [by weight] of anaerobic distilled water that contained 50 ,ug of deoxyribonuclease per ml) at 8,000 lb/in2 (563 kg/cm2) through a French pressure cell (American Instrument Co.), which was gassed with H2 before use. The disrupted cells were collected in a gassed centrifuge tube (1.4-cm ID), and the tube was sealed with a flanged hard stopper (1) and centrifuged under an H2 or N2 gas phase at 18,000 x g for 15 min. The supernatant fraction, which contained about 20 mg of protein per ml and approximately 0.2 ,umol of CO-oxidizing activity/min per mg of protein at 350C, was transferred with a syringe to anaerobic vials. A 105,000 x g supernatant fraction was prepared by centrifuging a 35,000 x g supernatant fraction under an H2 gas phase in sealed centrifuge tubes for 90 min at 105,000 x g in a Beckman ultracentrifuge (Spinco L2). Cell-free extracts of C. pasteurianum were prepared by incubating 2 g of frozen cells (wet weight) in 6 ml of anaerobic distilled water with 4 mg of lysozyme and 0.4 mg of deoxyribonuclease under H2 at 35°C for 30 min. The lysed cells were centrifuged at 35,000 x g for 15 min. The supernatant liquid contained about 30 mg of protein per ml and approximately 0.2 umol of CO-oxidizing activity/min per mg of protein at 35°C. For immediate use, the extracts were kept under strictly anaerobic conditions at 0°C. Under these conditions, 20 to 30% of the carbon monoxide-oxidizing activity was lost within 24 h. For later use, the extracts were stored at -200C under an H2, N2, or He gas phase. Protein was determined according to Lowry et al. (20) or Bradford (4), using bovine serum albumin as the standard. Preparation of F420. The procedure described by Tzeng et al. (37) was used for the isolation of F420

120

DANIELS ET AL.

from M. thermoautotrophicum. Visible spectra of oxidized and reduced F420 from M. thermoautotrophicum displayed spectral properties identical to those described for F2,, isolated from M. ruminantium (37) and Methanobacterium strain MOH (6). The spectral properties of F420 were analyzed by using a Gilford model 250 spectrophotometer equipped with a model 2030/35 wavelength scanner. Determination of metabolic gases. Carbon monoxide, CO2, CH4, and H2 were measured gas chromatographically on a Becker-Packard gas chromatograph 419 with thermal conductivity detection in combination with a two-channel recorder (OmniScribe, Houston Instruments). The operation conditions were as follows: stainless-steel column, 1.3 m by 0.32 cm, filled with Carbosieve B (120/140 mesh; Supelco Inc., Bellefonte, Pa.); He carrier gas (N2 when H2 was to be determined), 5 atm of pressure, 60 ml/min; temperatures - column, 100°C, detector, 125°C, and injection port, 45°C; filament current, 250 mA (using He as carrier gas) or 150 mA (using N2 as carrier gas). The CO concentration in aqueous solution at a given temperature was calculated from solubility data (31). CH4 alone was determined more sensitively, using a Varian Aerograph gas chromatograph 600-D with a flame ionization detector equipped with a stainless-steel column (1.5 m by 0.32 cm) filled with Porapak N (Waters Associates, Milford, Mass.) and heated at 75°C. N2 was used as the carrier gas at a flow rate of 30 ml/min at 1.5 atm of pressure. Gas samples of 0.2 and 0.4 ml were removed and injected by using a 1-ml GlasPak syringe (Becton, Dickinson and Co., Rutherford, N.J.) with a Teflon pressure lock device (Mininert syringe valve, Supelco Inc.) and a 23-gauge needle, both fitted tightly with Teflon tape. The radioactivity of '4C-labeled carbon monoxide, C02, and CH4 was determined as described previously (22) after separation and detection of the gases in the gas chromatograph with thermal conductivity detection. The outlet of the gas chromatograph column was connected to a Packard gas proportional counter (model 894). The concentrations and radioactivities of the gases were measured by relating the peak heights to a standard curve. Assay conditions for carbon monoxide oxidation in growing cultures. Carbon monoxide oxidation experiments with whole cells were conducted in 28-ml anaerobic tubes (aluminum seal tubes, Bellco Glass, Inc.) fitted with black rubber stoppers and secured with aluminum serum bottle seals (Wheaton Scientific) (1). Tubes that contained 9 ml of medium were inoculated with 1 ml of a growing culture and incubated horizontally at 65 or 30°C, at 60 oscillations/ min. In experiments with frozen cells, 1 ml of cell suspension was added instead of 1 ml of a growing culture. When carbon monoxide oxidation was tested with carbon monoxide as the sole energy source, a mixture of carbon monoxide and He (1 atm) was used as the gas phase. When carbon monoxide oxidation was to be measured in the presence of H2 and C02, it was assured that an equal amount of H2 and CO2 was present in all tubes, especially when the carbon monoxide concentration in the gas

J. BACTERIOL.

phase was varied. This was accomplished by adding 2 atm of overpressure of a gas mixture of 80% HJ 20% CO2 to the high-pressure tubes, which were already filled with a mixture of carbon monoxide and He at 1 atm of pressure. Conditions deviating from the standard procedure are given in the legends to figures and footnotes to the table. Assay conditions for carbon monoxide oxidation in cell-free extracts. For measuring the carbon monoxide-oxidizing activity in vitro, two methods were used. When the stoichiometry of CO oxidation was to be determined, the formation of "4CO2 from '4CO was measured gas chromatographically. Vials (7 ml) that contained 2 ml of test solution (100 mM potassium phosphate [pH 7.51-2 mM methyl viologen) were closed with rubber bungs and were evacuated and refilled several times with He to achieve anaerobic conditions. A 10 mM dithionite solution was added (4 to 50 AM) until the test solution turned slightly blue due to the reduction of the viologen dye, indicating completely anaerobic conditions. The "4C-labeled carbon monoxide was added with a gas-tight syringe. The reaction was started with 100 ,ul of extract, and the vials were incubated at 350C with shaking. After different times the reaction was stopped by injecting 0.4 ml of 40% trichloroacetic acid, and the vials were vigorously shaken to equilibrate "4CO2 in solution with the gas phase. Then, 0.4-ml gas samples were withdrawn with a gas-tight syringe and analyzed for "4CO and "4CO2. For all other in vitro investigations the carbon monoxide-oxidizing activity was determined by photometric determination of the reduction of methyl viologen or of F420 (see below) with carbon monoxide at 578 and 420 nm, respectively. The activity corresponds to the change in absorbance directly after initiating the reaction by adding extract to the test. One unit of enzyme activity is defined as 1 jAmol of carbon monoxide oxidized per min or 2 Zmol of methyl viologen reduced by carbon monoxide per min at 35°C with 100 mM potassium phosphate buffer, pH 7.5, according to the following equation: CO + H20 + 2 methyl viologeng+ - CO2 + 2H+ + 2 methyl viologen+d (e57, for methyl viologen = 9.7 mM- * cm-') The test used was specific for the carbon monoxideoxidizing activity. The assays were conducted in 1ml cuvettes with ground-glass fittings closed with soft-rubber stoppers. Anaerobic conditions were obtained by alternate evacuation and gassing of the cuvettes with CO or a CO/He mixture while shaking and by then adding enough (5 to 40 AM) dithionite to turn the test solution slightly blue. Then the cuvettes were warmed to the test temperature, and the gas phase and the liquid phase were equilibrated by vigorous shaking. The reaction was started by injecting cell-free extract or by changing the gas phase from CO to He, when the dependence of the reaction on CO was to be shown. The assay conditions for the reduction of F420 by CO were identical to those described above except that 2 mM dithioerythritol instead of dithionite was used as antioxidant and the A420 was monitored.

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121

F420, an electron acceptor of yet unknown structure, exhibits an absorption maximum at 420 nm in the oxidized state, in contrast to the reduced leukoform (6, 37). An F420 solution with 1 mg of F42O(0,) per ml has been reported to have a AA420 of 24 at a pH of 8.2 (37). LLJ

RESULTS The findings of Kluyver and Schnellen (19) that cell suspensions of two methanogenic bacteria removed carbon monoxide have been extended to growing cultures of different methanogens growing on H2/CO2 as energy source in the presence of 6% CO in the gas phase. M. thermoautotrophicum, M. barkeri, M. formicicum, M. ruminantium, M. arbophilicum, and Methanosarcina UBS removed CO from the gas phase at a rate of at least 10 ,mol of CO depleted/4 days per 10 ml of culture (containing approximately 5 mg [wet weight] of cells). Uninoculated controls showed that the loss of CO from the culture tube was dependent on the presence of cells. In addition, cell-free extracts of M. thermoautotrophicum, M. arbophilicum, and Methanosarcina UBS were shown to catalyze the oxidation of CO with methyl viologen as electron acceptor at a rate of approximately 0.2 ,umol/min per mg of protein (pH 7.5, 35°C). CO consumption by M. thermoautotrophicum growing on CO2 plus H2 in the presence of CO. Growth of M. thermoautotrophicum on C02 plus H2 as energy source was not significantly affected by CO concentrations up to 10% CO in the gas phase. However, higher CO concentrations were strongly inhibitory. At 25% CO in the gas phase, the rate of growth (A = 0.25/h, 2.8-h generation time) and the maximal rate of methane formation (1.2 ,umol/min per 10 ml of culture) were almost halved; at 40%o CO only 10% of the rates observed in the absence of CO were obtained. CO also had an effect on the lag phase, which was prolonged at higher CO concentrations. The inhibition of growth and methane formation by CO was reversible; after removal of CO, both growth and CH4 formation increased again to normal rates. In the experiments concerning CO consumption, growth, CO, and methane concentrations in the gas phase were followed from the moment of inoculation until the late stationary phase (Fig. 1A). CO consumption did not start until the late exponential phase, reached a maximum in the stationary phase when most cells were present, and continued for a number of hours after growth on C02 plus H2 and methane formation from C02 and H2 had virtually ceased. The rate of CO consumption by growing cultures of M. thermoautotrophicum, determined as indicated in Fig. 1A, was dependent

(I)

CD (0

I

a-

(D

UI) 0 -r

I

LUJ

I

I-

0

z 0 (-)

TIME ( hours )

1' L-

0 C

Co

EE

1

0 z

-

-

z 0 : 0 0

CO IN THE GAS PHASE((%/) FIG. 1. CO consumption by M, thermoautotrophicum growing on H2 plus C02 as energy source. (A) Growth and CO consumption as a function of time; (B) rate of CO consumption (slope A) as a function of the CO concentration initially present in the gas phase. The inset shows a double-reciprocal plot of the same data. [CO]0.5v, 10%; Vmaz, 210 nmol/min per10 ml of culture. Conditions: 10 ml ofa growing culture in a 28-ml sealed tube was continuously equilibrated at 65'C with the gas phase that contained a mixture of H2/CO2 (5:1; 2 atm and CO [amount indicated]). At the beginning of the stationary phase 10 ml of culture contained about 0.7 mg of cell protein.

122

DANIELS ET AL.

J. BACTERIOL.

tion present in the gas phase initially. The abscissa refers to the initial concentration of CO, whereas V was determined at a time when ~~~~~~25the CO concentration had already decreased. 02 The inset gives a reciprocal plot of the same ua E data, showing that at 10% CO (corresponding to 50 ,IM CO dissolved at 65°C) the rate is 50%o of 0 the extrapolated maximal rate of 210 nmol/min per 10 ml of culture. The extrapolated maximal 0 30 415 rate and the apparent Km for CO are only of operational interest, because growth and CH4 0 X~~~~~~~formation were increasingly inhibited at concen~ U S I0E1H trations higher than 10% CO in the gas phase. Growth of M. thermoautotrophicum on CO as sole energy source. M. thermoautotrophic 4 32 cum was found to grow on CO as sole energy 4!51 source (Fig. 2A). When cells were inoculated into a bicarbonate-buffered mineral medium that was continuously equilibrated with CO, TIME (HOURS) the cell density continuously increased. CO consumption and methane formation paralleled growth. A stoichiometry of approximately 4CO 0 ~~~~~~~~~~~~~~( disproportionated to 3CO2 and 1CH4 formed was observed (also see Table 1). Both growth (O)-h oethan 0~~~~~~~~~~~~~~~0 and methane fonnation were completely dependent on CO; it was established by gas chromatographic analysis that the CO used con) 0m tained less than 0.005% H2. Growth and CH4 formation were not observed in control cultures that lacked CO. The maximal growth rate on 0~~~~~~~~~~01 m -o CO was only 1% of that observed in H2CO2 as CE source. M. thermoautotrophicum was energy time Cotrl Osoednihrgot CDhu maintained and transferred many times for several months on a mineral medium that con2

co

20

OinO

0

.1

0

LU

(~~~~~~~0

0

0

w

U-

z

0

15

CO N Ii

30

THE GAS

45

60

PHASE ()m)

Growth

of M. thermoautotrophicum on CO as sole energy source. (A) Growth, CO depletion, and methane and CO2 as a function of time. Controls without CO showed neither growth nor CH, CO2 formation. (B) Growth (A) and methane (0) formation after 170 h as a function of the CO concentration in the gas phase. Conditions: (A) 1.0 ml of a culture growing on CO2 plus H2 (early a mineral phase) was added to 7.0 salts medium in 28-ml sealed tubes and continuously equilibrated at 650C with a ga-s phase that contained FIG.

2.

TABLE 1. Products formed from CO by growing cultures of M. thermoautotrophicum and by cell suspensions previously frozen at -20°C

iLmol/min per g of wet cells Cell source

formation

and

ml of

exponential

30% CO and He (1

atm). CO, represents total CO2/

HCO3- in the culture tube. (B) 0.5 ml of a culture growing on CO2 plus H2 (early exponential phase) was added to 9.5 ml of mineral salts medium in 28ml sealed tubes and continuously equilibrated at 650C with

atm) on

as9

a

gas

phase

that contained CO and He (1

indicated.

the CO concentration present in the gas

phase initially. Figure lB shows a plot of the rate of CO consumption versus CO concentra-

CO con-

CH,

formed

H2 formed

C02

formeda

sumed

Growing culturesb

9.4 2.1 tr 6.3 1.8 0.04 1.9 Cell suspensionc 2.0 trace 0.23 Cell suspension + 0.2 0.23 chloroform (2 dl/ Inl)_ _ a C02 was calculated from the amount of reduced products (CH4 and H2) formed. b 100 ml of a culture of M. thermoautotrophicum (20 mg of wet cells) actively growing on CO2 and H2 (early exponential phase) was added to 720 ml of mineral salts medium in a 920-ml Roux bottle and incubated at 65C under continuous shaking. The gas phase was 15% CO and 85% helium. The amounts of CO consumed and products formed were determined after 12 h. c Frozen wet cells of M. thermoautotrophicum (0.25 g) were added to 12.5 ml of mineral salts medium in a 28-ml sealed tube and incubated at 65°C under continuous shaking. The gas phase was 15% CO and 85% helium. The amounts of CO consumed and CH4 and H2 formed were determined after 1 h.

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CO OXIDATION BY METHANOGENIC BACTERIA

tained 30% CO in the gas phase as the sole energy source. A 10% inoculum was used in the transfer experinents and cells routinely grew to a cell density of about 107 cells per ml. The >10-fold increase in cell number observed after each transfer was completely dependent on the presence of CO, again indicating that CO sustained growth. Growth on CO and methane formation were dependent on the CO concentration (Fig. 2B). Both increased parallel with increasing CO concentration up to 30% in the gas phase and then decreased again to become virtually zero at concentrations higher than 60%. CO oxidation to CO2 with F420 as electron acceptor. The finding that 3 mol of C02 and 1 mol of CH4 are formed from 4 mol of CO during growth of M. thermoautotrophicum on CO clearly indicates that the methanogen must contain a CO dehydrogenase mediating the oxidation of CO to CO2. Primary information as to the electron acceptor was obtained by studying the utilization of CO by cells previously frozen at -20°C. These cells produced 1 mol of C02 and 1 mol of H2 for every 1 mol of CO used (Table 1). Only small amounts of CH4 were formed under these conditions. In the presence of chloroforn the formation of CH4 was completely abolished and only C02 and H2 were found as the end products. As the hydrogenase of methanogenic bacteria including M. thermoautotrophicum has been shown to be dependent on F420 (37; J. G. Zeikus, G. Fuchs, W. Kenealy, and R. K. Thauer, J. Bacteriol., in press), the formation of H2 from CO suggests that F,20 is somehow involved and might function as electron acceptor in CO oxidation. This was confirmed in studies with cell-free extracts of M. thermoautotrophicum. Cell-free extracts of this organism were shown to catalyze a rapid reduction of F420 with CO (Fig. 3). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and ferredoxin from C. pasteurianum were not reduced. Fornate could not replace CO in F420 reduction. Half-maximal velocity was observed at an F120 concentration equivalent to a A420 of 0.15. Cell-free extracts of C. pasteurianum did not reduce F,20 with CO (Fig. 3). Properties of the CO dehydrogenase activity. Extracts catalyzing the reduction of F420 with CO were also found to mediate the reduction of methyl and benzyl viologen with CO. A 2-mol amount of methyl viologen was shown to be reduced by 1 mol of CO with the concomitant formation of 1 mol of C02. Using these dyes as electron acceptors, the kinetics of CO oxidation and some properties of the CO-dehydrogenating enzyme were investigated. The reduction of

methyl viologen with CO proceeded linearly with time (0 to 4 min), and the rate of methyl viologen reduction was proportional to the amount of protein added (0 to 1 mg) in the range measurable photometrically (AA578 < 2). At 350C, pH 7.5, and with a CO concentration of 100% CO in the gas phase, a specific rate of 0.4 ,mmol of methyl viologen reduced/min per mg of protein was observed. The rate of methyl viologen reduction was linearly dependent on the carbon monoxide concentration with 0 and 100% CO in the gas phase (corresponding to 0 to 0.8 mM CO dissolved at 3500), indicating that the [S]0.5v for CO was higher than 1 mM. The dependence on the methyl viologen concentration was hyperbolic (not shown). From reciprocal plots, and [S]0.5V for methyl viologen of 20 ,mM was obtained. The rate of methyl viologen reduction with CO increased with increasing pH, with an inflection point at pH 6.7. The data fit reasonably well to a theoretical dissociation curve of a group with a pK of 6.7, indicating that the dissociation of a group with a pK of 6.7 (e.g., a histidine residue) might be rate limiting for the enzymatic methyl viologen reduction with CO.

0

-4

z

0 P-

C-) 0

LLJ

0

5

10

15

20

TIME (min) FIG. 3. F420 reduction with CO by cell-free extracts of M. thermoautotrophicum. Conditions: TricinelK+ buffer, pH 8.1, 100 mM; dithioerythritol, 2 mM; F420 as indicated by the absorbance; cell-free extract of M. thermoautotrophicum (0) or C. pasteurianum (A) as indicated, 5 pi; total volume, 1 ml; 40°C. The gas phase was changed from N2 to CO as indicated.

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DANIELS ET AL.

The temperature dependence of the rate followed the Arrhenius equation. From the data, a Q1,oC of 2.3 and an energy of activation of 14.1 kcal/mol (59.0 kJ/mol) was calculated (pH 7.5). The CO dehydrogenase of M. thermoautotrophicumis a soluble enzyme. Almost 100% of the activity of cell-free extracts was retained in the 105,000 x g supernatant fraction. The CO dehydrogenase was precipitated with 60% ammonium sulfate and was inactivated within minutes by heat (100°C), by alkaline pH (pH 12), and by acidic pH (pH 4). The activity was not lost by anaerobic dialysis against 10 mM phosphate buffer. The enzyme was rapidly inactivated by 02, 2 mM Hg+, and 5 mMp-chloromercuribenzoate. Incubation of the extracts with cyanide resulted in g loss of activity. The inactivation process followed first-order kinetics with an inactivation rate constant of 2 x 103 s-' m (350C, pH 7.5). The inactivation rate was not affected by CO (100% in the gas phase), and the inactivation was reversible; after removal of the cyanide the activity reappeared to 100% of the initial activity within a few hours. Other monovalent anions known to be ligands to transition metals, including fluoride, iodide, azide, iodate, thiocyanate, sulfite (each 5 mM), or complexing agents like ethylenediaminetetraacetic acid (5 mM) were without effect on the CO-oxidizing activity or the stability of the enzyme.

DISCUSSION In this communication we have confirmed that many methanogenic bacteria metabolize carbon monoxide and have demonstrated that M. thermoautotrophicum can grow on CO as sole energy source. Four moles of CO is disproportionated to 3 mol of CO2 and 1 mol of CH, under these conditions. The data presented that cell-free extracts of M. thermoautotrophicum contain an active CO dehydrogenase and a hydrogenase (Zeikus et al., in press) both specific for F420 as electron acceptor give a biochemical basis for the understanding of this physiological phenomenon. CO + H20 + nF42,OX)

H2

+

CO2 + 2H+

+

nF420,rd)

nF420o)x, > 2H+ + nF420(Fd)

Reduced F420 is known to be involved as electron donor in CO2 reduction to CH4 (6, 36, 37); CO2 reduction to methane with H2 is the en-

ergy-yielding process when methanogens grow on H2/CO2. In this case, F420 is reduced by hydrogen via hydrogenase (37); when they grow on CO as sole energy source (CO2 was always present in the medium), CO instead of H2 is

used

as

the electron donor via the F420-specific

CO dehydrogenase. CO and H2 differ, however, as electron donors for F,20 in that the specific rate of F,20 reduction with H2 (Zeikus et al., in press) is by a factor of 100 higher (7.7 gmol of H2 oxidized/min per mg of protein at 35°C) than with CO (0.06 ,umol of CO oxidized/min per mg of protein at 30% CO and 350C). The different rates sufficiently explain why the growth rate on CO was only about 1% of the rate on C02 plus H2. A maximal growth rate with CO of 1% of the rate observed with H2/CO2 means the doubling time is extremely slow with CO as the sole energy source. Although growth (as measured by CO-dependent increase in optical density) on CO is repeatable, the growth observed may differ from the usual meaning of bacterial growth that initiates with one cell. The possibility exists that only a part of the population might be viable when grown with CO alone. A nongrowing function of CO metabolism could be its conversion to H2 and C02, as in the case of resting cells, and the living part of the population would then outgrow on the H2 and CO2 produced. However, a more direct alternative explanation is that slowly growing cells are metabolizing CO and using the products of this reaction for growth. It is important to note that growth of M. thermoautotrophicum on CO can only be observed when the CO concentrations in the gas phase are not too high. At concentrations higher than 30% the growth rate drastically decreased, to become virtually zero at concentrations higher than 60%. The biochemical basis for the inhibition of growth on CO is not understood. The concentration of CO at which CO inhibition predominates over growth on CO may differ from organism to organism. Therefore, different CO concentrations must be tested before it can be determined whether a methanogen can grow on CO or not. The CO dehydrogenase of M. thermoautotrophicum exhibits many similarities with the metalloenzyme previously described for C. pasteurianum (34). Both enzymes catalyze the reduction of viologen dyes with CO and are reversibly inactivated by cyanide. Both enzymes are extremely oxygen sensitive and can be inactivated by mercurials. However, the enzymes differ in that the CO dehydrogenase from M. thermoautotrophicum is specific for F420 as physiological electron acceptor, whereas the clostridial enzyme does not mediate the reduction of F420 with CO. Quantitative differences are found in the [S]O..V values for methyl viologen (C. pasteurianum, 0.4 mM; M. thermoautotrophicum, 20 AM) and CO (C. pasteurianum, 5 AM; M. thermoautotrophicum, >1

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CO OXIDATION BY METHANOGENIC BACTERIA

mM) and the rates of cyanide inactivation (C. pasteurianum, k = 102 s-1 *m1; M. thermoautotrophicum, k = 2 x 103 -1. m-1). The much higher apparent Km value of the enzyme from M. thermoautotrophicum for CO and the high affinity to cyanide explains why cyanide inactivation of the methanogen enzyme was not affected by the presence of C0, whereas the clostridial enzyme was protected from cyanide inactivation by its substrate, CO. A difference was also observed in the dependence of the rate of methyl viologen reduction by CO on the pH. Whereas the pH activity curve of the clostridial dehydrogenase resembles a titration curve of a weak acid with a pK of 8.4 (34), that of the dehydrogenase of M. thermoautotrophicum was identical to a titration curve of a weak acid with a pK of 6.7. CO dehydrogenase, but not hydrogenase, was inactivated by cyanide, indicating that separate activities catalyze the oxidation of H2 and CO. Hydrogenase activity was inhibited by CO. Although hydrogen cannot definitely be excluded as an intermediate in methyl viologen reduction with C0, this does not seem very likely because the kinetics of methyl viologen reduction with CO are not in favor of the accumulation of an intermediate. In C. pasteurianum an involvement of H2 as an intermediate was excluded by showing that purified CO dehydrogenase mediates the reduction of viologen dyes in the complete absence of hydrogenase (unpublished observation).

Indirect evidence is available that the clostridial enzyme contains a transition metal as a prosthetic group which exhibits properties similar to cobalt in vitamin B12; e.g., the enzyme was shown to be inactivated by methyl iodide and to be reactivated by irradiation with the light of a projection lamp (34). CO oxidation to C02 and H2 was shown to be "inhibited" by chloroform (Table 1), as is C02 reduction to methane in methanogens (2, 26). The effect exerted by CHC13 on methane formation has been attributed to the formation of an alkylB12. The findings that the demethylation of methyl coenzyme M is inhibited by chloroform (21) and that the enzyme preparation mediating this reaction does not appear to contain a corrinoid (12a) suggest that the inhibition experiment cannot be interpreted unambiguously. C0 oxidation to C02 has been reported for a variety of different bacteria, both anaerobic and aerobic, including sulfate-reducing bacteria (25, 41, 42), clostridia (12, 32, 34), photosynthetic bacteria (13, 38), methylotrophic bacteria (7, 14), and H2-oxidizing bacteria (17, 18, 23, 24, 27, 28, 43). A hydrogen-oxidizing bacterium (28) and a photosynthetic bacterium (38) have been

125

reported to grow fairly well on C0. The mechanism of carbon monoxide oxidation appears not to be uniforn. Anaerobic bacteria and the hydrogen-oxidizing bacteria catalyze the oxidation of carbon monoxide to C02 via a dehydrogenase (34, 42; E. Helmke, thesis, Technische UniversitAt zu Braunschweig, Brunswick, West Germany, 1973), and the second 0 in C02 is derived from water. However, methane-oxidizing bacteria mediate the reaction via a monooxygenase and the second oxygen in C02 is derived from 02 (7, 8). In methane-oxidizing bacteria the carbon monoxide activity has been shown to be identical to the methane monooxygenase complex; i.e., the oxidation of carbon monoxide to C02 is not the physiological function of the enzyme. The same probably holds true for the carbon monoxide dehydrogenase of methanogenic bacteria, clostridia, and sulfatereducing bacteria (11). The physiological function of the carbon monoxide-oxidizing enzyme in these bacteria still remains to be established. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Science, University of Wisconsin, Madison; by grant DEB 73-01511-AO1 from the National Science Foundation to J.G.Z.; by a grant of the Deutsche Forschungsgemeinschaft to G.F.; and by project SFB103 in Marburg to R.K.T. The technical assistance of S. Klevickis in maintaining methanogenic cultures is appreciated. LITERATURE CITED 1. Balch, W. E., and R. S. Wolfe. 1976. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol. 32:781-791. 2. Bauchop, T. 1967. Inhibition of rumen methanogenesis by methane analogues. J. Bacteriol. 94:171-175. 3. Bidwell, R. G., and G. P. Bebee. 1974. Carbon monoxide fixation by plants. Can. J. Bot. 52:1841-1847. 4. Bradford, M. M. 1976. A rapid and sensitive method for

the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 5. Bryant, M. P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324-1328. 6. Cheeseman, P., A. Toms-Wood, and R. S. Wolfe. 1972. Isolation and properties of a fluorescent compound, factors,0, from Methanobacterium strain M.o.H. J. Bacteriol. 112:527-531. 7. Ferenci, T. 1974. Carbon monoxide stimulated respiration in methane-utilizing bacteria. FEBS Lett. 41:9498. 8. Ferenci, T. 1977. Carbon monoxide oxidation by methane-utilizing bacteria. In Proceedings of Symposium on Microbial Production and Utilization of Gases

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11. Fuchs, G., G. Andrew., and R. K. Thauer. 1977. CO-