JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4464 4471 VOl. 172, NO. 8

0021-9193/90/084464-08$02.00/0Copyright © 1990, American Society for Microbiology

Characterization of the H2- and CO-Dependent ChemolithotrophicPotentials of the Acetogens Clostridium thermoaceticum

and Acetogenium kivuitSTEVEN L. DANIEL, TSUNGDA HSU, SARA I. DEAN, AND HAROLD L. DRAKE*

Microbial Physiology Laboratories, Department of Biology, The Universityof Mississippi, University, Mississippi 38677

Received 22 January 1990/Accepted 18 May 1990

Strains of Clostidum thermoaceticum were tested for H2- and CO-dependent growth in a defined mediumcontaining metals, minerals, vitamins, cysteine-sulfide, C02-bicarbonate, and H2 or CO. Ten of the thirteenstrains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for furtherstudy. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditiods (the definedmedium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively.Product stoichiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2-+CH3COOH + 0.1 cellC + 0.3 unrecovered C and 6.8CO-*CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependentgrowth produced signifiantly higher acetate concentrations per unit of biomass synthesized than did CO- or

glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophicconditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 valus for A. kivuiand C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the Ycovalue for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of

hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source ufflized for growthand were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells andbenzyl viologen as electron acceptor, the V,. values for hydrogenase from C. thermoaceticum and A. kivuiwere 155.7 and 1,670 ,umol of H2 oxidized per min per mg of protein, respectively; the V__ values for COdehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 ,umol of CO oxidized per min per mg

of protein, respectively.

In 1936, the first acetogen, Clostridium aceticum, wasisolated (55); however, the culture was lost and, until re-cently reisolated (4), unavailable for study. Clostridiumthermoaceticum was isolated in 1942 (14). Unlike C. aceti-cum, which grows at the expense of H2-CO2, C. thermoace-ticum was isolated as a strict heterotroph. For many years,C. thermoaceticum was the only acetogen available forstudy, and physiological and enzymological studies over thepast 40 years with this organism have been pivotal inelucidation of the autotrophic acetyl coenzyme A (acetyl-CoA) or Wood pathway (35, 56). As pointed out by Wood etal. (56), it was somewhat peculiar that an obligate hetero-troph possessed this autotrophic pathway. However, theoccurrence of hydrogenase (10) and CO dehydrogenase (9,12) in this organism, as well as the ability of the organism toutilize H2-CO2 or CO as a carbon and energy (reductant)source for acetogenesis under heterotrophic conditions (e.g.,glucose- or yeast extract-enriched environments; 26-28, 43),suggests that C. thermoaceticum is also capable of au-totrophic growth and acetogenesis.

Besides acetogens, acetogenesis is also a fundamentalbiological process to the autotrophic metabolism of metha-nogens and sulfate-reducing bacteria (15, 35, 56, 61). Giventhe cornerstone role C. thermoaceticum has played in reso-lution of the acetyl-CoA pathway, we have recently eluci-

* Corresponding author.t Dedicated to Harland G. Wood, whose pioneering work with

Clostridium thermoaceticum led to the discovery of a new au-totrophic pathway, the acetyl coenzyme A (Wood) pathway.

dated the minimal nutritional requirements of this acetogen(37) so that a definitive assessment could be made of itsheterotrophic and chemolithotrophic potentials. In the studypresented here, numerous strains of C. thermoaceticumwere obtained from various sources and evaluated. In addi-tion, Acetogenium kivui (33, 34), a thermophilic nonclostrid-ial acetogen which is capable of H2-dependent chemo-lithotrophic growth, was also included in this evaluation andused for comparative purposes. In this report, we demon-strate for the first time that certain strains of C. thermoace-ticum grow chemolithotrophically (requiring only trace lev-els of nicotinic acid as the sole vitamin) at the expense of H2or CO. Besides the overall metabolic properties exhibited byC. thermoaceticum and A. kivui under chemolithotrophicconditions, evidence is also presented which suggests thatthe type of energy source used during growth (e.g., H2versus glucose) influences the expression or activity ofhydrogenase and CO dehydrogenase in both of these aceto-gens.

MATERIALS AND METHODS

Bacterial strains and cultivation. C. thermoaceticum (seeTable 1 for strains used in this study) and A. kivui ATCC33488 were cultivated at 55°C in butyl rubber-stopperedcrimp-sealed culture tubes (18 by 150 mm; series 2048[Bellco Glass, Inc., Vineland, N.J.]; 27.2-ml approximatestoppered volume at 1 atm [101.29 kPa]). The undefinedmedium contained, in milligrams per liter: NaHCO3, 3,500;KH2PO4, 500; NaCl, 400; NH4Cl, 400; MgCl2 6H20, 330;CaCl2 2H20, 50; resazurin, 1; yeast extract, 1,000; nico-

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TABLE 1. Growth of C. thermoaceticum at the expense of glucose, methanol, CO, and H2

Maximum growth (A6.)bStrain designationa; Undefined medium Defined mediumsource

Glucose Methanol CO H2 Glucose Methanol CO H2Wood;H. Wood ++++ +++ + + +++ ++ ++ +Barker;L. Ljungdahl +++ ++ ++ + +++ +++ ++ +OMD;H. Drake ++++ - - - ++++- - -OML;H. Drake +++ - + - +++ - ++Methanoladapted;J. Wiegel ++++ +++++ +++ ++ ++++ ++ +++ +Ljungdahl; J. Wiegel ++++ ++++ +++ ++ ++++ ++ +++ +CO adapted; J. Zeikus ++++ +++ + + +++ ++ ++ +Fontaine;J. Zeikus ++++ +++ ++ + +++ ++ ++ +DSM2955;J. Andreesen +++++ +++ ++ + ++++ ++ ++ +SG;D. Wang ++++ - - - +++ATCC 39289; CPC International +++ ++++ + ++ ++ +ATCC31490;ATCC +++ +++ ++ + +++ + +ATCC 39073; ATCC +++++ +++ ++ + ++++ ++ ++ +

a ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen.b Measured after a minimum of three sequential passages in the undefined or defined medium with either 10 mM glucose, 60 mM methanol, 30% CO, or 30%H2 as the energy source. Inocula for undefined and defined media were from undefined and defined glucose medium cultures, respectively. Scale: -, no growthobserved (240 h of incubation) after first or second passage; +, <0.2; ++, 0.2 to 0.4; +++, 0.41 to 0.6; ++++, 0.61 to 0.8; +++++, >0.8. Values werecorrected for inoculum.

tinic acid, 0.25; cyanocobalamin, 0.25; p-aminobenzoi'c acid,0.25; calcium D-pantothenate, 0.25; thiamine * HCI, 0.25;riboflavin, 0.25; lipoic acid, 0.15; folic acid, 0.1; biotin, 0.1;pyridoxal- HCl, 0.05; sodium nitrilotriacetate, 7.5; MnSO4 -H20, 2.5; FeSO4 *7H20, 0.5; Co(NO3)2 * 6H20, 0.5; ZnCl2,0.5; NiCl2 - 6H20, 0.25, CuS04 - 5H20, 0.05; AlK(S04)2 -12H20, 0.05; H3BO3, 0.05; and Na2MoO4 - 2H20, 0.05. Theundefined medium was prepared anaerobically by boilingand cooling the medium under 100% C02, by dispensing themedium under 100% N2 into culture tubes (7 ml per tube),and by adding sodium sulfide-cysteine solution (0.07 ml/tube[46]); culture tubes were subsequently crimp sealed andautoclaved. The defined medium was the undefined mediumwithout yeast extract. The minimal medium was the definedmedium with nicotinic acid as the sole vitamin and withsodium sulfide (at twice the concentration) as the solereducing agent (cysteine and NaOH omitted from solution).The basal medium was the minimal medium without nico-tinic acid.

Before inoculation, culture tubes were pressurized atroom temperature to a pressure of 140 kPa (10 lb/in2 overatmospheric pressure) with 100% CO2; unless indicatedotherwise, this was the initial cultivation gas phase (CO2-N2[40:60]). The initial pH of culture media approximated 6.5.When growth was at the expense of glucose or methanol,these substrates were added to media at final concentrationsof 10 and 60 mM, respectively, before autoclaving. Whengrowth was at the expense of CO or H2, culture tubes werepressurized at room temperature to a final total pressure of240 kPa (20 lb/in2 over atmospheric pressure) with either100% CO or H2; the cultivation gas phase contained CO-C02-N2 or H2-CO2-N2 (30:30:40). All gases were passedover a copper catalyst at 450°C to remove trace amounts ofoxygen and were filter sterilized before introduction intoculture tubes. In all experiments, growth was initiated byinjecting 0.5 ml of inoculum per culture tube, and culturetubes were incubated horizontally (without shaking).

Analytical procedures. Growth was quantitated at 660 nmwith a Spectronic 501 spectrophotometer (Bausch & Lomb,Inc., Rochester, N.Y.); the optical path width (inner diam-eter of culture tubes) was 1.6 cm. Uninoculated mediumserved as a reference. Cell dry weights were determined as

previously described (38, 58). For product profile analyses,the carbon and hydrogen content of cells was assumed toapproximate 50 and 8% of the cell dry weight, respectively(48).

Substrates and end products in culture fluids were quan-titated with a high-performance liquid chromatograph(109OL; Hewlett-Packard Co., Palo Alto, Calif.) equippedwith a fermentation monitoring column (Bio-Rad Laborato-ries, Inc., Richmond, Calif.), UV (210 nm) and refractiveindex (1037A; Hewlett-Packard Co.) detectors, and an inte-grator (4290; Spectra-Physics, Bedford, Mass.). Chromato-graphic conditions were as follows: column oven, 60°C; 0.01N H2SO4 mobile phase at a flow rate of 0.8 ml/min; andinjection size, 10 p.1. Before chromatographic analysis, cul-ture fluids were clarified by microcentrifugation and micro-filtration.Headspace gases were quantitated with a gas-liquid chro-

matograph (5790A; Hewlett-Packard Co.) equipped withstainless steel columns (1.8 mm by 2 m) containing Molecu-lar Sieve (13 x 60/80 mesh; Supelco, Inc., Bellefonte, Pa.) forCO or H2 analysis and Chromosorb 102 (60/80 mesh; Su-pelco) for CO2 analysis, a thermal conductivity detector, andan integrator (3390A; Hewlett-Packard Co.). Chromato-graphic conditions were as follows: injection port, 150°C;column oven, 60°C; detector, 175°C; 100% argon carrier gasat a flow rate of 20 ml/min; and injection size, 50 ,ul. Beforechromatographic analysis, culture fluids in tubes were acid-ified with 0.5 ml of 12 N HCl; after chromatographic analy-sis, the volume of headspace gas was measured manometri-cally. CO, H2, and CO2 solubilities were calculated fromstandard solubility tables (53), and the amount of gas pro-duced or consumed was calculated by taking in account bothgas and liquid phases. CO, H2, and CO2 recoveries fromuninoculated tubes averaged 96, 99, and 92%, respectively.

Preparation of ceil extracts and enzyme assays. Cell ex-tracts of C. thermoaceticum ATCC 39073 and A. kivuiATCC 33488 were prepared in an anaerobic chamber (N2-H2[95:5]) by lysozyme digestion (37). Hydrogenase (10) and COdehydrogenase (11, 12) activities in extracts were assayed bystandard techniques with benzyl viologen as the electronacceptor, and the apparent Km and Vma values for CO andH2 were determined from Lineweaver-Burk plots. The gas

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phase for CO dehydrogenase and hydrogenase assays was100% H2 and CO (calculated soluble concentrations approx-imated 0.64 mM), respectively. Protein was estimated by themethod of Bradford (3).

Chemicals. All chemicals used were of the highest puritycommercially available. Metabolic inhibitors were obtainedfrom Sigma Chemical Co. (St. Louis, Mo.) or AldrichChemical Co., Inc. (Milwaukee, Wis.). Stock solutions ofmetabolic inhibitors were prepared in either 80% ethanol ordeionized water.

RESULTS

Screening strains of C. thermoaceticum for 112- and CO-dependent growth. To assess the autotrophic growth poten-tials of C. thermoaceticum, strains of this acetogen werescreened for H2- and CO-dependent growth in both unde-fined and defined media (Table 1). Of the 13 strains tested inthe undefined medium, 10 strains grew at the expense of H2and CO, one strain (OML) grew at the expense of only CO,and two strains (OMD and SG) failed to grow at the expenseof either H2 or CO; similar growth responses to H2 and COwere observed with these same strains in the defined me-dium (Table 1). Interestingly, both strains that did notdisplay either H2- or CO-dependent growth also failed togrow at the expense of methanol. Of the strains screened, C.thermoaceticum ATCC 39073 was chosen for further studyon the basis of the fact that it grew well on each of the foursubstrates tested and was commercially available.Undefined glucose medium cultures of C. thermoaceticum

ATCC 39073 yielded gram-positive (weakly) rod-shapedcells with very few spores when Gram stained. The generallack of spores by C. thermoaceticum supports previousfindings (54) and is in contrast to Clostridium thermoau-totrophicum, a closely related thermophilic acetogen (6),which sporulates more frequently (54). However, to excludethe possibility of a mixed (containing a strain of C. ther-moaceticum such as OMD which does not display H2- orCO-dependent growth) or contaminated culture, C. ther-moaceticum ATCC 39073 was reisolated from both regularand heat-shocked (100°C for 5 min) undefined glucose me-dium cultures on undefined glucose medium solidified with1% Gelrite. Colonies were predominantly small, circular,and white. All 22 (14 and 8 from regular and heat-shockedcultures, respectively) of the colonies tested grew at theexpense of glucose, methanol, CO, or H2 in the undefinedmedium (data not shown). In addition, undefined glucosemedium cultures did not grow when incubated at 25°C orwhen the undefined glucose medium (minus reducer andbicarbonate and supplemented with 0.5 g of K2HPO4 perliter of medium [pH adjusted to 6.5]) was prepared underaerobic conditions and incubated at 25 or 55°C. Also, C.thermoaceticum ATCC 39073, as well as all of the otherstrains in Table 1, was capable of glucose-dependent growthin the minimal medium; however, none of the strains grew inthe basal glucose medium (data not shown). These resultsagree with a previous report (37) that nicotinic acid isrequired for glucose-dependent growth of C. thermoaceti-cum. These findings also indicate the C. thermoaceticumATCC 39073 culture was pure.Growth profiles of C. thermoacelicum and A. kivui, H2- and

CO-dependent growth profiles of C. thermoaceticum ATCC39073 are shown in Fig. 1 and 2, respectively. With H2-cultivated cells, increasingly minimal growth conditions hadlittle, if any, effect on cell yields while doubling timesincreased (Fig. 1 and Table 2); with CO-cultivated cells, cell

0(0

30.02 80

0.01

0 40 80 120 160 200Cultivation Time (h)

FIG. 1. H2-dependent growth profiles of C. thermoaceticumATCC 39073 in media containing an initial cultivation gas phase ofH2-C02-N2 (30:30:40) at a total pressure of 240 kPa. Symbols: A,undefined medium; 0, defined medium; 0, defined medium withoutcysteine; A, minimal medium; *, basal medium; E, A. kivui ATCC33488 in basal medium.

yields decreased while doubling times remained nearly un-changed with increasingly minimal conditions (Fig. 2 andTable 2). In the minimal medium, the doubling times for cellscultivated chemolithotrophically with H2 or CO were 25 and10 h, respectively, whereas the doubling time for cellscultivated heterotrophically with glucose was 6 h (Table 2).

05

0.2

0.1

0~~~~

0

0.02 260 h

0.01 J

0 20 40 60 80 100Cultivation Time (h)

FIG. 2. CO-dependent growth profiles of C. thermoaceticumATCC 39073 in media containing an initial cultivation gas phase ofCO-CO2-N2 (30:30:40) at a total pressure of 240 kPa. Symbols: A,undefined medium; 0, defined medium; A, minimal medium; 0,basal medium; O, A. kivui ATCC 33488 in basal medium.

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TABLE 2. Doubling times and cell and acetate yields of C. thermoaceticum ATCC 39073 and A. kivui ATCC 33488a

Organism Medium Energy Passage Doubling Acetate Cell yield Acetate/cellsourceb no.c time (h) concn (mM) (g [dry wt]/liter) dry wt ratioC. thermoaceticum Undefined Glucose 31 4.0 10.8 0.21 51ATCC 39073 CO 22 10.0 7.6 0.17 45

H2 22 8.5 14.6 0.06 243Defined Glucose 30 6.0 13.1 0.20 66

CO 23 9.0 8.2 0.13 63H2 23 16.0 16.6 0.04 415

Minimal Glucose 23 6.0 6.4 0.07 91CO 14 10.0 8.4 0.10 84H2 5 25.0 21.0 0.05 420

A. kivui ATCC 33488 Basal Glucose 4 2.7 15.2 0.43 35H2 4 2.7 14.5 0.07 207

a Values are means of triplicate cultures.b Glucose, CO, and H2 concentrations were 10 mM, 30%1, and 30o, respectively.c Number of sequential passages on each medium.

No growth was observed in the minimal medium in theabsence of H2 or CO (data not shown) or in the basal mediumwith H2 or CO (Fig. 1 and 2), the latter indicating thatnicotinic acid was required for H2- or CO-dependent chem-olithotrophic growth of C. thermoaceticum.A. kivui ATCC 33488 has no essential vitamin requirement

and grows under chemolithotrophic conditions at the ex-pense of H2 (33). In this study, A. kivui grew in the basalmedium at the expense of H2 (Fig. 1). Growth did not occurin either the basal or the undefined medium in the absence ofH2 (data not shown). In addition, CO-dependent growth wasnot observed in the basal medium (Fig. 2), the undefinedmedium (data not shown), or the buffered medium (33; withor without yeast extract) used in the original isolation of thisorganism (data not shown). When compared during H2-dependent chemolithotrophic growth, the doubling time forA. kivui was 2.7 h, approximately 10-fold less than thedoubling time observed for C. thermoaceticum (Table 2).

Chemolithotrophic and heterotrophic product profiles.Based on substrate-to-product profiles of C. thermoaceticumATCC 39073 under chemolithotrophic conditions (Table 3),H2- and CO-dependent growth yielded the following stoichi-ometries, respectively:4.1H + 2.4CO2- CH3COOH + 0.1 cell C + 0.3 unrecovered C6.8COCH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered CGlucose-cultivated cells yielded the following stoichiometry(Table 3):

C6H1206 + 0.7C02j-2.7CH3COOH + 0.4 cell C + 0.9unrecovered C

Based on these stoichiometries, approximately 4, 6, and 7%

of the CO2 (in H2-CO2 cultures), CO, and glucose consumedwere accounted for in biomass carbon, respectively, underminimal conditions. Besides biomass, the sole product de-tected by H2-cultivated cells was acetate, whereas CO-cultivated cells formed not only acetate and a small amountof H2 (0.2 mM) (Table 3) but also an unidentified compound.The low hydrogen recovery observed with CO-cultivatedcells may be attributed in part to this unknown product;chromatographic and colorimetric (31) analyses indicatedthat it was not formate. In addition, this unidentified com-pound was also observed in culture fluids from CO-growncells ofPeptostreptococcus productus U-1 (data not shown).The major products detected from glucose-cultivated cells ofC. thermoaceticum (minimal medium; Table 3) and A. kivuiATCC 33488 (basal medium; data not shown) were biomassand acetate; H2 was a minor product (0.2 mM). In earlierstudies, H2 production by C. thermoaceticum was shown tooccur during CO- or glucose-dependent growth in complexundefined media (26, 42); this is the first report of H2production by A. kivui during growth at the expense ofglucose.

Effects of cultivation conditions on the bioenergetics ofgrowth. Since energy production and biomass synthesis areobligately coupled to acetogenesis, the ratio of acetateformed to biomass synthesized is a direct reflection of cellenergetics (27, 46, 47, 52). Overall, with increasingly mini-mal conditions, acetate-to-biomass ratios (the amount ofacetate produced per unit of biomass synthesized) of C.thermoaceticum ATCC 39073 increased (Table 2), therebydemonstrating that the cell experienced an increased energydemand under increasingly minimal conditions. Increased

TABLE 3. Glucose, CO, and H2 product profiles of C. thermoaceticum ATCC 39073a

Energy Substrate utilized" Product formed % Recoverysource Glucose CO H2 CO2 Acetate CO2 H2 Biomass Cc Biomass Hd Carbon Hydrogen

Glucose 9.4 NAe NA 6.2 25.0 -1 0.2 3.4 6.5 85 95CO NA 68.4 NA NA 10.1 35.6 0.2 3.7 7.0 87 70H2 NA NA 77.5 45.6 19.1 1.5 2.9 87 101

a Units are in micromoles per milliliter of culture; values are means of two or three experiments (triplicate cultures [incubated until maximum absorbance wasachieved] per experiment).bAt zero time, glucose, CO, H2, and CO2 concentrations in the minimal medium cultures averaged 9.4, 79.2, 78.5, and 114.5 ,umol/ml, respectively.c Assuming 50%0 carbon per unit of cell biomass, 1 mg of cell dry weight is equivalent to 41.6 F.mol of biomass carbon.d Assuming 8% hydrogen per unit of cell biomass, 1 mg of cell dry weight is equivalent to 80 ,umol of biomass hydrogen.NA, Not applicable.

f-, Not observed.

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TABLE 4. Effects of metabolic inhibitors on the growth ofC. thermoaceticum ATCC 39073

Inhibitor Growth (% of control)'(RM), Glucose Methanol Syringate CO H2

TBT (200) 100 0 0 0 0DCCD (500) 100 100 8 0 0DNP (200) 75 0 80 80 0CCCP (500) 57 0 0 83 NDcMonensin (10) 0 0 0 0 0Valinomycin (10) 8 ND 7 8 0Nigericin (10) 0 ND 0 0 NDAmiloride (200) 0 0 0 0 0Harmaline (200) 90 114 88 85 78KCN (200) 59 103 100 81 100

a TBT, Tributyltin chloride; DCCD, N,N'-dicyclohexylcarbodiimide;DNP, 2,4-dinitrophenol; CCCP, carbonyl cyanide m-chlorophenylhydrazone.

b Determined by measuring growth (A660) in the defined medium (with andwithout inhibitor) with either 10 mM glucose, 60 mM methanol, 10 mMsyringate, 30% CO, or 30%o H2 as the energy source.

c ND, Not determined.

acetate-to-biomass ratios relative to increasingly minimalgrowth conditions have also been observed with C. ther-moautotrophicum (46). In the minimal medium (Table 2), theacetate-to-biomass ratio for glucose-cultivated cells of C.thermoaceticum was about five times less than that ofH2-cultivated cells. A similar relationship was observedbetween the acetate-to-biomass ratios for glucose- and H2-cultivated cells ofA. kivui ATCC 33488 (Table 2). However,when compared during H2-dependent chemolithotrophicgrowth, the acetate-to-biomass ratio for A. kivui was slightlyless than half that of C. thermoaceticum.To further assess potential differences between au-

totrophic and heterotrophic acetogenesis, various metabolicinhibitors were examined for their effects on glucose-, meth-anol-, syringate-, CO-, and H2-dependent growth by C.thermoaceticum ATCC 39073 (Table 4). Chemolithotrophi-cally (H2 or CO) and heterotrophically (glucose) grown cellsdiplayed differential growth sensitivities to tributyltin chlo-ride and N,N'-dicyclohexylcarbodiimide, two putative AT-Pase inhibitors. In contrast, H2-, CO-, and glucose-depen-dent growth were equally inhibited by the metal ionophoresmonensin, valinomycin, and nigericin. Of the two putativeNa+/H+ antiporter inhibitors tested (amiloride and harma-line), only amiloride inhibited both chemolithotrophic andheterotrophic cells.

Effects of chemolithotrophic and heterotrophic growth sub-strates on the levels of hydrogenase and CO dehydrogenase.Hydrogenase and CO dehydrogenase are critical to au-totrophic acetogenesis. Overall, each of the growth sub-strates tested had some degree of influence on hydrogenaseand CO dehydrogenase activities in C. thermoaceticumATCC 39073 and A. kivui ATCC 33488 (Table 5). With C.thermoaceticum, H2-dependent chemolithotrophic cells con-tained the highest levels of hydrogenase and CO dehydroge-nase (based on Vm. values). In addition, the levels of thesetwo enzymes were significantly greater in A. kivui than in C.thermoaceticum.

DISCUSSION

This study demonstrates that C. thermoaceticum is capa-ble of H2- or CO-dependent growth and acetogenesis underchemolithotrophic conditions. Reasons for the apparent dif-ferences in autotrophic potentials among strains of C. ther-

TABLE 5. Hydrogenase and CO dehydrogenase activities in cellextracts of C. thermoaceticum ATCC 39073 and

A. kivui ATCC 33488

Enzyme activity'Energy CO dehydro-Organism sourcea Hydrogenase genase

Km Vmax Km VM

C. thermoaceticum Glucose 0.53 10.2 0.94 18.0ATCC 39073 Methanol 0.48 11.9 0.92 40.0

Syringate 1.08 28.8 0.83 34.4CO 1.54 8.8 2.62 40.7H2 0.16 155.7 0.29 90.6

A. kivui ATCC 33488 Glucose 0.26 550.0 0.24 2,733.0H2 0.44 1,670.0 0.35 2,973.0

aEnergy sources were glucose (10 mM), methanol (60 mM), syringate (10mM), CO (30%o), and H2 (30%) in the defined medium.

b K,,,, Micromolar benzyl viologen; Vm., micromoles ofH2 or CO oxidizedper minute per milligram of cell extract protein (benzyl viologen as electronacceptor).

moaceticum are not known (Table 1); however, since allstrains have been derived from the original isolate of Fon-taine et al. (14), some strains have apparently lost thecapacity for autotrophic growth.To the best of our knowledge, A. kivui ATCC 33488 has

the fastest growth rate of any known acetogen. However, A.kivui appears to be incapable ofgrowth at the sole expense ofCO, and the inability to grow at the sole expense of CO isapparently not due to a lack of CO dehydrogenase. Indeed,supplemental CO is consumed by A. kivui during H2- orglucose-dependent growth and stimulates both cell and ace-tate yields of H2-cultivated cells (59). Two possibilities mayexplain why A. kivui is unable to grow at the sole expense ofCO: (i) the acetyl-CoA pathway is altered in a manner whichprevents the total synthesis of acetyl-CoA from CO, or (ii)the utilization of CO is not effectively coupled to energyconservation or anabolic processes. Similar metabolic limi-tations may also explain the loss of autotrophic potentials bycertain strains of C. thermoaceticum (e.g., strain OMD,which contains hydrogenase [10] and CO dehydrogenase [11]and utilizes H2 and CO during glucose-dependent growth[26, 27, 43] but is unable to grow autotrophically).The mechanism(s) by which autotrophically grown aceto-

gens conserve energy has not been evaluated. From studiesprimarily with heterotrophically grown acetogens, the fol-lowing facts are known about the bioenergetics of acetogens:(i) growth yields suggest the involvement of electron trans-port phosphorylation in energy conservation (1, 19, 35); (ii)acetogens are rich in metalloenzymes and electron carriers(7, 20, 35, 45, 60); (iii) an ATPase and other catalysts, whichare components of a membrane-associated electron trans-port system, have been identified in C. thermoaceticum andC. thermoautotrophicum (7, 23-25, 44); and (iv) sodium is animportant element in energy conservation of some acetogens(17, 21, 50, 59). However, whether the mechanism(s) ofenergy conservation during autotrophic and heterotrophicgrowth is the same remains unclear.

In this study, autotrophically and heterotrophically growncells of C. thermoaceticum ATCC 39073 displayed differen-tial growth sensitivities to metabolic inhibitors, especiallythe putative inhibitors of ATPase tributyltin chloride andN,N'-dicyclohexylcarbodiimide. Similarly, the growth andenergy-dependent transport of nickel by H2- and glucose-grown cells ofA. kivui also display differential sensitivities toATPase inhibitors (58). Sodium is essential for the H2-

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CHEMOLITHOTROPHIC POTENTIALS OF C. THERMOACETICUM 4469

TABLE 6. Comparative growth yields for H2- or CO-grown acetogensaAcetogen (strain) Medium YH2 Yco Reference

Acetobacterium woodii (NZva 16) Defined 0.68b NRc 52Acetobacterium carbinolicum (DSM 2925) Defined 0.675 NR 13Acetobacterium malicum (DSM 4132) Defined 1.17 NR 49Acetogenium kivui (ATCC 33488) Basal 0.91 _d This studyAOR Undefined 0.28b +e 32"Butyribacterium methylotrophicum" Undefined 1.7 3.0 40, 41Clostridium thermoaceticum (ATCC 39073) Minimal 0.46 1.28 This studyClostridium thermoautotrophicum (JW701/3) Defined 0.82 2.53 46, 47Clostridium pfennigii (DSM 3222) Undefined - 2.50f 30Eubacterium limosum (RF) Undefined 0.84 3.38 18Peptostreptococcus productus (ATCC 35244) Undefined 0.65f 2.13f 36P. productus (Marburg) Undefined + 3.2 16Sporomusa termitida (DSM 4440) Undefined 0.50 + 5

a Unless otherwise indicated, units are grams of cell dry weight per mole of H2 or CO consumed.bOriginal data reported as grams of cell dry weight per mole of acetate formed. The values shown were derived by dividing by 4 (see reaction in Discussion).c NR, Not reported; whether or not CO supports growth was also not reported.d -, Substrate did not support growth.+, Substrate did support growth; however, growth yield was not reported.

f Original data reported as grams of cell protein per mole of substrate (H2 or CO) consumed. The values shown were calculated on the basis of the assumptionthat cells were 60% protein (30).

dependent growth of A. kivui; however, glucose-dependentgrowth does not depend on supplemental sodium (59). Sim-ilar responses to sodium have been reported for Acetobac-terium woodii (21). In contrast, C. thermoaceticum is sensi-tive to metal ionophores and the putative Na+/H+ antiporterinhibitor amiloride (Table 4) but does not display any depen-dence on supplemental sodium (59). The differential re-sponses to metabolic inhibitors and sodium suggest that themechanisms of energy conservation during autotrophic andheterotrophic growth may not be the same for some aceto-gens.The overall theoretical stoichiometries and the standard

changes in Gibbs free energy for H2- and CO-derived au-totrophic acetogenesis are, respectively:

4H2 + 2C02CH3COOH + 2H20 (-23.4 kJ/mol H2; 51)4CO + 2H2OCH3COOH + 2CO2 (-41.4 kJ/mol CO; 36)

Both forms of autotrophic acetogenesis require 8 reducingequivalents for the synthesis of acetate. However, Yco isconsistently greater than YH2 for acetogens (Table 6). Inaddition, the acetate-to-biomass ratios for CO-grown cellsare substantially lower than those of H2-grown cells (Table2). The differences in H2- and CO-dependent growth effi-ciencies may be a result of (i) increased synthesis ofATP perCO-derived electron pair and (ii) the necessity to form COfrom C02, an energy-requiring process (8), during H2-depen-dent acetogenesis. However, since ATP is still required forthe synthesis of formyltetrahydrofolate from CO (27), no netincrease in ATP yields can be envisioned from substrate-level phosphorylation (via acetate kinase [35]) during CO-derived acetogenesis. It seems likely that the same is trueduring H2-derived acetogenesis. Thus, both H2- and CO-dependent growth would appear to be strictly dependent onelectron transport phosphorylation for the conservation ofchemolithotrophically derived energy.By virtue of its central role in acetogenesis (35, 56), CO

dehydrogenase (acetyl-CoA synthetase) is clearly a criticalenzyme under both heterotrophic and chemolithotrophicconditions. In contrast, while hydrogenase provides the cellwith utilizable energy and reductant during H2-dependentacetogenesis, the physiological role this enzyme plays duringheterotrophic acetogenesis remains unknown. In this study,cells of C. thermoaceticum ATCC 39073 grown chemo-

lithotrophically at the expense of H2 had higher levels ofthese enzymes than did cells grown at the expense of CO orheterotrophic substrates (Table 5). Hydrogenase levels werealso greater in chemolithotrophic (H2-grown) cells ofA. kivuiATCC 33488. Hydrogenase and CO dehydrogenase activi-ties in A. kivui are the highest of any known acetogen (1,670and 2,973 pLmol of H2 and CO oxidized per min per mg ofextract protein from H2-grown cells, respectively). In com-parison, hydrogenase and CO dehydrogenase activities inextracts of CO-grown cells of "B. methylotrophicum" CO-adapted strain (29, 40; methyl viologen as electron acceptor),H2-grown cells of C. thermoaceticum ATCC 39073 (Table 5),and H2-grown cells of C. thermoautotrophicum DSM 1974(6; methyl viologen as electron acceptor) were 0.9 and 13.3,155.7 and 90.6, and 0.31 and 10.7 ,umol of H2 and COoxidized per min per mg of extract protein, respectively; COdehydrogenase activity in CO-grown cells of P. productusMarburg was 0.5 pumol of CO oxidized per min per mg ofextract protein (2).

Recently, two new inducible catalytic activities, an 0-demethylating enzyme system (57) and an aromatic decar-boxylating enzyme system (22; T. Hsu and H. L. Drake,submitted for publication), have been described in C. ther-moaceticum. Furthermore, C. thermoaceticum has the ca-pacity to transform aromatic aldehyde groups (39). Thesenew findings, together with the fact that C. thermoaceticumis capable of chemolithotrophic growth, serve to illustratethe diverse catabolic and anabolic potentials of this aceto-gen. Given the historical role that C. thermoaceticum hasplayed in establishing the acetyl-CoA pathway as a funda-mental autotrophic process, these recent developments in-dicate that this acetogen will continue to be an importantmodel for further resolving the overall impact and biochem-istry of acetogenesis.

ACKNOWLEDGMENTSWe express appreciation to all of the individuals listed in Table 1

who sent us cultures of C. thermoaceticum.This investigation was supported by Public Health Service grant

A121852 and research career development award A100722 (H.L.D.)from the National Institute of Allergy and Infectious Diseases, by aPublic Health Service biomedical research support grant to theUniversity of Mississippi from the National Institutes of Health, and

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4470 DANIEL ET AL.

by an award from the Associates' Funds from the University ofMississippi.

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