8 JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4464 4471 VOl. 172, NO. 0021-9193/90/084464-08$02.00/0 Copyright © 1990, American Society for Microbiology Characterization of the H2- and CO-Dependent Chemolithotrophic Potentials of the Clostridium thermoaceticum and Acetogenium kivuit STEVEN L. DANIEL, TSUNGDA HSU, SARA I. DEAN, AND HAROLD L. DRAKE* Microbial Physiology Laboratories, Department ofBiology, The University ofMississippi, 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 medium containing metals, minerals, vitamins, cysteine-sulfide, C02-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditiods (the defined medium 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 cell C + 0.3 unrecovered C and 6.8CO-*CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced signifiantly higher concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 valus for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the Yco value 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 growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the V,. values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 ,umol of H2 oxidized per min per mg of protein, respectively; the V__ values for CO dehydrogenase 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 , Clostridium aceticum, was dated the minimal nutritional requirements of this acetogen isolated (55); however, the culture was lost and, until re- (37) so that a definitive assessment could be made of its cently reisolated (4), unavailable for study. Clostridium heterotrophic and chemolithotrophic potentials. In the study thermoaceticum was isolated in 1942 (14). Unlike C. aceti- presented here, numerous strains of C. thermoaceticum cum, which grows at the expense of H2-CO2, C. thermoace- were obtained from various sources and evaluated. In addi- ticum was isolated as a strict heterotroph. For many years, tion, Acetogenium kivui (33, 34), a thermophilic nonclostrid- C. thermoaceticum was the only acetogen available for ial acetogen which is capable of H2-dependent chemo- study, and physiological and enzymological studies over the lithotrophic growth, was also included in this evaluation and past 40 years with this organism have been pivotal in used for comparative purposes. In this report, we demon- coenzyme A (acetyl- strate for the first time that certain strains of C. thermoace- elucidation of the autotrophic acetyl lev- CoA) or Wood pathway (35, 56). As pointed out by Wood et ticum grow chemolithotrophically (requiring only trace al. (56), it was somewhat peculiar that an obligate hetero- els of nicotinic acid as the sole vitamin) at the expense of H2 troph possessed this autotrophic pathway. However, the or CO. Besides the overall metabolic properties exhibited by occurrence of hydrogenase (10) and CO dehydrogenase (9, C. thermoaceticum and A. kivui under chemolithotrophic 12) in this organism, as well as the ability of the organism to conditions, evidence is also presented which suggests that energy (reductant) the type of energy source used during growth (e.g., H2 utilize H2-CO2 or CO as a carbon and of source for acetogenesis under heterotrophic conditions (e.g., versus glucose) influences the expression or activity glucose- or yeast extract-enriched environments; 26-28, 43), hydrogenase and CO dehydrogenase in both of these aceto- suggests that C. thermoaceticum is also capable of au- gens. totrophic growth and acetogenesis. Besides acetogens, acetogenesis is also a fundamental MATERIALS AND METHODS metha- biological process to the autotrophic of Bacterial strains and cultivation. C. thermoaceticum (see nogens and sulfate-reducing (15, 35, 56, 61). Given and A. kivui ATCC played in reso- Table 1 for strains used in this study) the cornerstone role C. thermoaceticum has 33488 were cultivated at 55°C in butyl rubber-stoppered lution of the acetyl-CoA pathway, we have recently eluci- crimp-sealed culture tubes (18 by 150 mm; series 2048 [Bellco Glass, Inc., Vineland, N.J.]; 27.2-ml approximate stoppered volume at 1 atm [101.29 kPa]). The undefined * Corresponding author. in milligrams per liter: NaHCO3, 3,500; to G. whose pioneering work with medium contained, t Dedicated Harland Wood, KH2PO4, 500; NaCl, 400; NH4Cl, 400; MgCl2 6H20, 330; Clostridium thermoaceticum led to the discovery of a new au- nico- totrophic pathway, the acetyl coenzyme A (Wood) pathway. CaCl2 2H20, 50; resazurin, 1; yeast extract, 1,000; 4464 VOL. 172, 1990 CHEMOLITHOTROPHIC POTENTIALS OF C. THERMOACETICUM 4465

TABLE 1. Growth of C. thermoaceticum at the expense of glucose, methanol, CO, and H2 Maximum growth (A6.)b Strain designationa; Undefined source medium Defined medium Glucose Methanol CO H2 Glucose Methanol CO H2 Wood;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; b DSM, Deutsche Sammlung von Mikroorganismen. Measured after a minimum of three sequential passages in the undefined or defined medium with either 10 mM H2 as the energy source. glucose, 60 mM methanol, 30% CO, or 30% Inocula for undefined and defined media were from undefined and defined glucose medium cultures, respectively. Scale: -, no growth observed (240 h of incubation) after first or second passage; +, <0.2; ++, 0.2 to 0.4; +++, 0.41 to 0.6; ++++, 0.61 corrected for inoculum. to 0.8; +++++, >0.8. Values were

tinic acid, 0.25; cyanocobalamin, 0.25; p-aminobenzoi'c acid, described For 0.25; calcium previously (38, 58). product profile analyses, D-pantothenate, 0.25; thiamine * HCI, 0.25; the carbon and hydrogen content of cells was assumed to riboflavin, 0.25; lipoic acid, 0.15; folic acid, 0.1; biotin, 0.1; approximate 50 and 8% of the cell dry weight, respectively pyridoxal- HCl, 0.05; sodium nitrilotriacetate, 7.5; MnSO4 - (48). H20, 2.5; FeSO4 *7H20, 0.5; Co(NO3)2 * 6H20, 0.5; ZnCl2, Substrates and end products in culture fluids were quan- - 0.5; NiCl2 - 6H20, 0.25, CuS04 5H20, 0.05; AlK(S04)2 - titated with a high-performance liquid chromatograph 12H20, 0.05; H3BO3, 0.05; and Na2MoO4 - 2H20, 0.05. The (109OL; Hewlett-Packard Co., Palo Alto, Calif.) equipped undefined medium was prepared anaerobically by boiling with a monitoring column (Bio-Rad Laborato- and cooling the medium under 100% C02, by dispensing the ries, Inc., Richmond, UV and medium under 100% Calif.), (210 nm) refractive N2 into culture tubes (7 ml per tube), index (1037A; Hewlett-Packard Co.) detectors, and an inte- and by adding sodium sulfide-cysteine solution (0.07 ml/tube grator (4290; [46]); culture tubes Spectra-Physics, Bedford, Mass.). Chromato- were subsequently crimp sealed and graphic conditions were as follows: column oven, 60°C; 0.01 autoclaved. The defined medium was the undefined medium N mobile without H2SO4 phase at a flow rate of 0.8 ml/min; and yeast extract. The minimal medium was the defined injection size, 10 p.1. Before chromatographic analysis, cul- medium with nicotinic acid as the sole vitamin and with ture fluids were clarified by microcentrifugation and micro- sodium sulfide (at twice the concentration) as the sole filtration. reducing agent (cysteine and NaOH omitted from solution). Headspace gases were quantitated with a chro- The basal medium was the minimal medium without nico- gas-liquid tinic acid. matograph (5790A; Hewlett-Packard Co.) equipped with stainless steel columns (1.8 mm by 2 m) containing Molecu- Before inoculation, culture tubes were pressurized at lar Sieve (13 x 60/80 mesh; Supelco, Inc., Bellefonte, Pa.) for room temperature to a pressure of 140 kPa (10 lb/in2 over CO or and Chromosorb 102 atmospheric 100% H2 analysis (60/80 mesh; Su- pressure) with CO2; unless indicated pelco) for CO2 analysis, a thermal conductivity detector, and otherwise, this was the initial cultivation gas phase (CO2-N2 an integrator (3390A; Hewlett-Packard Co.). Chromato- [40:60]). The initial pH of culture media approximated 6.5. graphic conditions were as follows: When growth was at the injection port, 150°C; expense of glucose or methanol, column oven, 60°C; detector, 175°C; 100% argon carrier gas these substrates were added to media at final concentrations at a flow rate of 20 and of 10 and 60 ml/min; injection size, 50 ,ul. Before mM, respectively, before autoclaving. When chromatographic analysis, culture fluids in tubes were acid- growth was at the expense of CO or H2, culture tubes were ified with 0.5 ml of 12 N HCl; after chromatographic analy- pressurized at room temperature to a final total pressure of sis, the volume of was 240 kPa (20 headspace gas measured manometri- lb/in2 over atmospheric pressure) with either cally. CO, H2, and CO2 solubilities were calculated from 100% CO or H2; the cultivation gas phase contained CO- standard tables and the C02-N2 or solubility (53), amount of gas pro- H2-CO2-N2 (30:30:40). All gases were passed duced or consumed was calculated by taking in account both over a copper catalyst at 450°C to remove trace amounts of gas and liquid phases. CO, H2, and CO2 recoveries from oxygen and were filter sterilized before introduction into uninoculated tubes and culture tubes. In all experiments, averaged 96, 99, 92%, respectively. growth was initiated by Preparation of ceil extracts and enzyme assays. Cell ex- injecting 0.5 ml of inoculum per culture tube, and culture tracts of C. tubes were thermoaceticum ATCC 39073 and A. kivui incubated horizontally (without shaking). ATCC 33488 were prepared in an anaerobic chamber Analytical procedures. Growth was quantitated at 660 nm (N2-H2 with a [95:5]) by lysozyme digestion (37). Hydrogenase (10) and CO Spectronic 501 spectrophotometer (Bausch & Lomb, dehydrogenase (11, activities in extracts were Inc., Rochester, N.Y.); the 12) assayed by optical path width (inner diam- standard techniques with benzyl viologen as the electron eter of culture tubes) was 1.6 cm. Uninoculated medium acceptor, and the and served as a reference. Cell apparent Km Vma values for CO and dry weights were determined as H2 were determined from Lineweaver-Burk plots. The gas 4466 DANIEL ET AL. J. BACTERIOL. phase for CO dehydrogenase and hydrogenase assays was 100% H2 and CO (calculated soluble concentrations approx- imated 0.64 mM), respectively. Protein was estimated by the method of Bradford (3). Chemicals. All chemicals used were of the highest purity commercially available. Metabolic inhibitors were obtained from Sigma Chemical Co. (St. Louis, Mo.) or Aldrich 0 Chemical Co., Inc. (Milwaukee, Wis.). Stock solutions of (0 metabolic inhibitors were prepared in either 80% ethanol or deionized water. 30.02 RESULTS 8 0 Screening strains of C. thermoaceticum for 112- and CO- dependent growth. To assess the autotrophic growth poten- 0.01 tials of C. thermoaceticum, strains of this acetogen were screened for H2- and CO-dependent growth in both unde- fined and defined media (Table 1). Of the 13 strains tested in the undefined medium, 10 strains grew at the expense of H2 and CO, one strain (OML) grew at the expense of only CO, 0 40 80 120 160 200 and two strains (OMD and SG) failed to grow at the expense Cultivation Time (h) of either H2 or CO; similar growth responses to H2 and CO were observed with these same strains in the defined me- FIG. 1. H2-dependent growth profiles of C. thermoaceticum dium (Table 1). Interestingly, both strains that did not ATCC 39073 in media containing an initial cultivation gas phase of display either H2- or H2-C02-N2 (30:30:40) at a total pressure of 240 kPa. Symbols: A, CO-dependent growth also failed to undefined medium; 0, defined medium; 0, defined medium without grow at the expense of methanol. Ofthe strains screened, C. cysteine; A, minimal medium; *, basal medium; E, A. kivui ATCC thermoaceticum ATCC 39073 was chosen for further study 33488 in basal medium. on the basis of the fact that it grew well on each of the four substrates tested and was commercially available. Undefined glucose medium cultures of C. thermoaceticum yields decreased while doubling times remained nearly un- ATCC 39073 yielded gram-positive (weakly) rod-shaped changed with increasingly minimal conditions (Fig. 2 and cells with very few spores when Gram stained. The general Table 2). In the minimal medium, the doubling times for cells lack of spores by C. thermoaceticum supports previous cultivated chemolithotrophically with H2 or CO were 25 and findings (54) and is in contrast to Clostridium thermoau- 10 h, respectively, whereas the doubling time for cells totrophicum, a closely related thermophilic acetogen (6), cultivated heterotrophically with glucose was 6 h (Table 2). which sporulates more frequently (54). However, to exclude the possibility of a mixed (containing a strain of C. ther- moaceticum such as OMD which does not display H2- or CO-dependent growth) or contaminated culture, C. ther- 05 moaceticum ATCC 39073 was reisolated from both regular and heat-shocked (100°C for 5 min) undefined glucose me- dium cultures on undefined glucose medium solidified with 1% Gelrite. Colonies were predominantly small, circular, 0.2 and white. All 22 (14 and 8 from regular and heat-shocked cultures, respectively) of the colonies tested grew at the expense of glucose, methanol, CO, or H2 in the undefined 0.1 medium (data not shown). In addition, undefined glucose medium cultures did not grow when incubated at 25°C or when the undefined glucose medium (minus reducer and 0~~~~ bicarbonate and supplemented with 0.5 g of K2HPO4 per liter of medium [pH adjusted to 6.5]) was prepared under aerobic conditions and incubated at 25 or 55°C. Also, C. 0 thermoaceticum ATCC 39073, as well as all of the other strains in Table 1, was capable of glucose-dependent growth 0.02 in the minimal medium; however, none of the strains grew in 260 h the basal glucose medium (data not shown). These results agree with a previous report (37) that nicotinic acid is required for glucose-dependent growth of C. thermoaceti- 0.01 J cum. These findings also indicate the C. thermoaceticum ATCC 39073 culture was pure. 0 20 40 60 80 100 Growth profiles of C. thermoacelicum and A. kivui, H2- and Cultivation Time (h) CO-dependent growth profiles of C. thermoaceticum ATCC FIG. 2. CO-dependent growth profiles of C. thermoaceticum 39073 are shown in Fig. 1 and 2, respectively. With H2- ATCC 39073 in media containing an initial cultivation gas phase of cultivated cells, increasingly minimal growth conditions had CO-CO2-N2 (30:30:40) at a total pressure of 240 kPa. Symbols: A, little, if any, effect on cell yields while doubling times undefined medium; 0, defined medium; A, minimal medium; 0, increased (Fig. 1 and Table 2); with CO-cultivated cells, cell basal medium; O, A. kivui ATCC 33488 in basal medium. VOL. 172, 1990 CHEMOLITHOTROPHIC POTENTIALS OF C. THERMOACETICUM 4467

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 sourceb no.c time (h) concn (mM) (g [dry wt]/liter) dryAcetate/cellwt ratio C. thermoaceticum Undefined Glucose 31 4.0 10.8 0.21 51 ATCC 39073 CO 22 10.0 7.6 0.17 45 H2 22 8.5 14.6 0.06 243 Defined Glucose 30 6.0 13.1 0.20 66 CO 23 9.0 8.2 0.13 63 H2 23 16.0 16.6 0.04 415 Minimal Glucose 23 6.0 6.4 0.07 91 CO 14 10.0 8.4 0.10 84 H2 5 25.0 21.0 0.05 420 A. kivui ATCC 33488 Basal Glucose 4 2.7 15.2 0.43 35 H2 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 the of the CO2 (in H2-CO2 cultures), CO, and glucose consumed absence of H2 or CO (data not shown) or in the basal medium were accounted for in biomass carbon, respectively, under with H2 or CO (Fig. 1 and 2), the latter indicating that minimal conditions. Besides biomass, the sole product de- nicotinic acid was required for H2- or CO-dependent chem- tected by H2-cultivated cells was acetate, whereas CO- olithotrophic growth of C. thermoaceticum. cultivated cells formed not only acetate and a small amount A. kivui ATCC no 33488 has essential vitamin requirement of H2 (0.2 mM) (Table 3) but also an unidentified compound. and grows under chemolithotrophic conditions at the ex- The low hydrogen recovery observed with CO-cultivated pense of H2 (33). In this study, A. kivui grew in the basal cells may be attributed in part to this unknown product; medium at the expense of H2 (Fig. 1). Growth did not occur chromatographic and colorimetric analyses indicated in either the basal or (31) the undefined medium in the absence of that it was not formate. In this H2 (data not shown). In addition, unidentified com- addition, CO-dependent growth was pound was also observed in culture fluids from not observed in the basal medium (Fig. CO-grown 2), the undefined cells ofPeptostreptococcus productus U-1 (data not shown). medium (data not shown), or the buffered medium (33; with The detected from or without yeast extract) used in the major products glucose-cultivated cells of original isolation of this C. thermoaceticum (minimal medium; Table 3) and A. kivui organism (data not shown). When compared during H2- ATCC 33488 dependent chemolithotrophic growth, the doubling time for (basal medium; data not shown) were biomass and was a minor A. kivui was 2.7 h, approximately 10-fold less than the acetate; H2 product (0.2 mM). In earlier studies, H2 production by C. thermoaceticum was shown to doubling time observed for C. thermoaceticum (Table 2). Chemolithotrophic and heterotrophic product profiles. occur during CO- or glucose-dependent growth in complex Based on substrate-to-product profiles of C. thermoaceticum undefined media (26, 42); this is the first report of H2 ATCC 39073 under chemolithotrophic conditions (Table 3), production by A. kivui during growth at the expense of H2- and CO-dependent growth yielded the following stoichi- glucose. ometries, respectively: Effects of cultivation conditions on the bioenergetics of growth. Since energy production and biomass synthesis are 4.1H + 2.4CO2- CH3COOH + 0.1 cell C + 0.3 unrecovered C obligately coupled to acetogenesis, the ratio of acetate 6.8COCH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C formed to biomass synthesized is a direct reflection of cell Glucose-cultivated cells yielded the following stoichiometry energetics (27, 46, 47, 52). Overall, with increasingly mini- (Table 3): mal conditions, acetate-to-biomass ratios (the amount of acetate produced per unit of biomass synthesized) of C. C6H1206 + 0.7C02j-2.7CH3COOH + 0.4 cell C + 0.9 unrecovered C thermoaceticum ATCC 39073 increased (Table 2), thereby demonstrating that the cell experienced an increased energy Based on these stoichiometries, approximately 4, 6, and 7% demand under increasingly minimal conditions. Increased

TABLE 3. Glucose, CO, and H2 product profiles of C. thermoaceticum ATCC 39073a Energy Substrate utilized" Product formed % Recovery source 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 95 CO NA 68.4 NA NA 10.1 35.6 0.2 3.7 7.0 87 70 H2 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 was achieved] 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. 4468 DANIEL ET AL. J. BACTERIOL.

TABLE 4. Effects of metabolic inhibitors on the growth of TABLE 5. Hydrogenase and CO dehydrogenase activities in cell C. thermoaceticum ATCC 39073 extracts of C. thermoaceticum ATCC 39073 and A. kivui ATCC 33488 Inhibitor Growth (% of control)' (RM), Glucose Methanol Syringate CO H2 Enzyme activity' Energy CO dehydro- TBT (200) 100 0 0 0 0 Organism sourcea Hydrogenase genase DCCD (500) 100 100 8 0 0 DNP (200) 75 0 80 80 0 Km Vmax Km VM CCCP (500) 57 0 0 83 NDc Monensin (10) 0 0 0 0 0 C. thermoaceticum Glucose 0.53 10.2 0.94 18.0 Valinomycin (10) 8 ND 7 8 0 ATCC 39073 Methanol 0.48 11.9 0.92 40.0 Nigericin (10) 0 ND 0 0 ND Syringate 1.08 28.8 0.83 34.4 Amiloride (200) 0 0 0 0 0 CO 1.54 8.8 2.62 40.7 Harmaline (200) 90 114 88 85 78 H2 0.16 155.7 0.29 90.6 KCN (200) 59 103 100 A. kivui ATCC 33488 Glucose 0.26 550.0 0.24 2,733.0 81 100 H2 0.44 1,670.0 0.35 2,973.0 a TBT, Tributyltin chloride; DCCD, N,N'-dicyclohexylcarbodiimide; DNP, 2,4-dinitrophenol; CCCP, carbonyl cyanide m-chlorophenylhydrazone. aEnergy sources were glucose (10 mM), methanol (60 mM), syringate (10 b Determined by measuring growth (A660) in the defined medium (with and mM), CO (30%o), and H2 (30%) in the defined medium. without inhibitor) with either 10 mM glucose, 60 mM methanol, 10 mM b K,,,, Micromolar benzyl viologen; Vm., micromoles ofH2 or CO oxidized syringate, 30% CO, or 30%o H2 as the energy source. per minute per milligram of cell extract protein (benzyl viologen as electron c ND, Not determined. acceptor). moaceticum are not known (Table 1); however, since all acetate-to-biomass ratios relative to increasingly minimal strains have been derived from the original isolate of Fon- growth conditions have also been observed with C. ther- taine et al. (14), some strains have apparently lost the moautotrophicum (46). In the minimal medium (Table 2), the capacity for autotrophic growth. acetate-to-biomass ratio for glucose-cultivated cells of C. To the best of our knowledge, A. kivui ATCC 33488 has thermoaceticum was about five times less than that of the fastest growth rate of any known acetogen. However, A. H2-cultivated cells. A similar relationship was observed kivui appears to be incapable ofgrowth at the sole expense of between the acetate-to-biomass ratios for glucose- and H2- CO, and the inability to grow at the sole expense of CO is cultivated cells ofA. kivui ATCC 33488 (Table 2). However, apparently not due to a lack of CO dehydrogenase. Indeed, when compared during H2-dependent chemolithotrophic supplemental CO is consumed by A. kivui during H2- or growth, the acetate-to-biomass ratio for A. kivui was slightly glucose-dependent growth and stimulates both cell and ace- less than half that of C. thermoaceticum. tate yields of H2-cultivated cells (59). Two possibilities may To further assess potential differences between au- explain why A. kivui is unable to grow at the sole expense of totrophic and heterotrophic acetogenesis, various metabolic CO: (i) the acetyl-CoA pathway is altered in a manner which inhibitors were examined for their effects on glucose-, meth- prevents the total synthesis of acetyl-CoA from CO, or (ii) anol-, syringate-, CO-, and H2-dependent growth by C. the utilization of CO is not effectively coupled to energy thermoaceticum ATCC 39073 (Table 4). Chemolithotrophi- conservation or anabolic processes. Similar metabolic limi- cally (H2 or CO) and heterotrophically (glucose) grown cells tations may also explain the loss of autotrophic potentials by diplayed differential growth sensitivities to tributyltin chlo- certain strains of C. thermoaceticum (e.g., strain OMD, ride and N,N'-dicyclohexylcarbodiimide, two putative AT- which contains hydrogenase [10] and CO dehydrogenase [11] Pase inhibitors. In contrast, H2-, CO-, and glucose-depen- and utilizes H2 and CO during glucose-dependent growth dent growth were equally inhibited by the metal ionophores [26, 27, 43] but is unable to grow autotrophically). monensin, valinomycin, and nigericin. Of the two putative The mechanism(s) by which autotrophically grown aceto- Na+/H+ antiporter inhibitors tested (amiloride and harma- gens conserve energy has not been evaluated. From studies line), only amiloride inhibited both chemolithotrophic and primarily with heterotrophically grown acetogens, the fol- heterotrophic cells. lowing facts are known about the bioenergetics of acetogens: Effects of chemolithotrophic and heterotrophic growth sub- (i) growth yields suggest the involvement of electron trans- strates on the levels of hydrogenase and CO dehydrogenase. port phosphorylation in energy conservation (1, 19, 35); (ii) Hydrogenase and CO dehydrogenase are critical to au- acetogens are rich in metalloenzymes and electron carriers totrophic acetogenesis. Overall, each of the growth sub- (7, 20, 35, 45, 60); (iii) an ATPase and other catalysts, which strates tested had some degree of influence on hydrogenase are components of a membrane-associated electron trans- and CO dehydrogenase activities in C. thermoaceticum port system, have been identified in C. thermoaceticum and ATCC 39073 and A. kivui ATCC 33488 (Table 5). With C. C. thermoautotrophicum (7, 23-25, 44); and (iv) sodium is an thermoaceticum, H2-dependent chemolithotrophic cells con- important element in energy conservation of some acetogens tained the highest levels of hydrogenase and CO dehydroge- (17, 21, 50, 59). However, whether the mechanism(s) of nase (based on Vm. values). In addition, the levels of these energy conservation during autotrophic and heterotrophic two enzymes were significantly greater in A. kivui than in C. growth is the same remains unclear. thermoaceticum. In this study, autotrophically and heterotrophically grown cells of C. thermoaceticum ATCC 39073 displayed differen- DISCUSSION tial growth sensitivities to metabolic inhibitors, especially the putative inhibitors of ATPase tributyltin chloride and This study demonstrates that C. thermoaceticum is capa- N,N'-dicyclohexylcarbodiimide. Similarly, the growth and ble of H2- or CO-dependent growth and acetogenesis under energy-dependent transport of nickel by H2- and glucose- chemolithotrophic conditions. Reasons for the apparent dif- grown cells ofA. kivui also display differential sensitivities to ferences in autotrophic potentials among strains of C. ther- ATPase inhibitors (58). Sodium is essential for the H2- VOL. 172, 1990 CHEMOLITHOTROPHIC POTENTIALS OF C. THERMOACETICUM 4469

TABLE 6. Comparative growth yields for H2- or CO-grown acetogensa Acetogen (strain) Medium YH2 Yco Reference Acetobacterium woodii (NZva 16) Defined 0.68b NRc 52 Acetobacterium carbinolicum (DSM 2925) Defined 0.675 NR 13 Acetobacterium malicum (DSM 4132) Defined 1.17 NR 49 Acetogenium kivui (ATCC 33488) Basal 0.91 _d This study AOR Undefined 0.28b +e 32 "Butyribacterium methylotrophicum" Undefined 1.7 3.0 40, 41 Clostridium thermoaceticum (ATCC 39073) Minimal 0.46 1.28 This study Clostridium thermoautotrophicum (JW701/3) Defined 0.82 2.53 46, 47 Clostridium pfennigii (DSM 3222) Undefined - 2.50f 30 Eubacterium limosum (RF) Undefined 0.84 3.38 18 Peptostreptococcus productus (ATCC 35244) Undefined 0.65f 2.13f 36 P. productus (Marburg) Undefined + 3.2 16 Sporomusa 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 assumption that cells were 60% protein (30).

dependent growth of A. kivui; however, glucose-dependent lithotrophically at the expense of H2 had higher levels of growth does not depend on supplemental sodium (59). Sim- these enzymes than did cells grown at the expense of CO or ilar responses to sodium have been reported for Acetobac- heterotrophic substrates (Table 5). Hydrogenase levels were terium woodii (21). In contrast, C. thermoaceticum is sensi- also greater in chemolithotrophic (H2-grown) cells ofA. kivui tive to metal ionophores and the putative Na+/H+ antiporter ATCC 33488. Hydrogenase and CO dehydrogenase activi- inhibitor amiloride (Table 4) but does not display any depen- ties in A. kivui are the highest of any known acetogen (1,670 dence on supplemental sodium (59). The differential re- and 2,973 pLmol of H2 and CO oxidized per min per mg of sponses to metabolic inhibitors and sodium suggest that the extract protein from H2-grown cells, respectively). In com- mechanisms of energy conservation during autotrophic and parison, hydrogenase and CO dehydrogenase activities in heterotrophic growth may not be the same for some aceto- extracts of CO-grown cells of "B. methylotrophicum" CO- gens. adapted strain (29, 40; methyl viologen as electron acceptor), The overall theoretical stoichiometries and the standard H2-grown cells of C. thermoaceticum ATCC 39073 (Table 5), changes in Gibbs free energy for H2- and CO-derived au- and H2-grown cells of C. thermoautotrophicum DSM 1974 totrophic acetogenesis are, respectively: (6; methyl viologen as electron acceptor) were 0.9 and 13.3, 155.7 4H2 + 2C02CH3COOH + 2H20 (-23.4 kJ/mol H2; 51) and 90.6, and 0.31 and 10.7 ,umol of H2 and CO 4CO + 2H2OCH3COOH + 2CO2 (-41.4 kJ/mol CO; 36) oxidized per min per mg of extract protein, respectively; CO Both forms of autotrophic dehydrogenase activity in CO-grown cells of P. productus acetogenesis require 8 reducing Marburg was 0.5 pumol of CO oxidized per min per mg of equivalents for the synthesis of acetate. However, Yco is extract consistently greater protein (2). than YH2 for acetogens (Table 6). In Recently, two new inducible an addition, the acetate-to-biomass ratios for CO-grown cells catalytic activities, 0- are substantially lower demethylating enzyme system (57) and an aromatic decar- than those of H2-grown cells (Table boxylating enzyme system (22; T. Hsu and H. L. Drake, 2). The differences in H2- and CO-dependent growth effi- submitted for have ciencies may be a result of (i) increased publication), been described in C. ther- synthesis of ATP per moaceticum. Furthermore, C. thermoaceticum has the ca- CO-derived electron pair and (ii) the necessity to form CO pacity to transform aromatic from C02, an energy-requiring process (8), during H2-depen- aldehyde groups (39). These dent acetogenesis. new findings, together with the fact that C. thermoaceticum However, since ATP is still required for is capable of chemolithotrophic serve to the synthesis of formyltetrahydrofolate from CO (27), no net growth, illustrate increase the diverse catabolic and anabolic potentials of this aceto- in ATP yields can be envisioned from substrate- gen. Given the historical role that C. level phosphorylation (via acetate kinase [35]) during CO- thermoaceticum has derived played in establishing the acetyl-CoA pathway as a funda- acetogenesis. It seems likely that the same is true mental autotrophic process, these recent during H2-derived acetogenesis. Thus, both H2- and CO- developments in- dependent dicate that this acetogen will continue to be an important growth would appear to be strictly dependent on model for further resolving the overall impact and biochem- electron transport phosphorylation for the conservation of of chemolithotrophically derived energy. istry acetogenesis. By virtue of its central role in acetogenesis (35, 56), CO ACKNOWLEDGMENTS dehydrogenase (acetyl-CoA synthetase) is clearly a critical enzyme under both heterotrophic and chemolithotrophic We express appreciation to all of the individuals listed in Table 1 conditions. In contrast, while hydrogenase provides the cell who sent us cultures of C. thermoaceticum. with utilizable This investigation was supported by Public Health Service grant energy and reductant during H2-dependent A121852 and acetogenesis, the physiological role this enzyme plays during research career development award A100722 (H.L.D.) from the National Institute of Allergy and Infectious Diseases, by a heterotrophic acetogenesis remains unknown. In this study, Public Health Service biomedical research support grant to the cells of C. thermoaceticum ATCC 39073 grown chemo- University ofMississippi from the National Institutes of Health, and 4470 DANIEL ET AL. J. BACTERIOL. by an award from the Associates' Funds from the University of 21. Heise, R., V. Muller, and G. Gottschalk. 1989. Sodium depen- Mississippi. dence of acetate formation by the acetogenic bacterium Aceto- bacterium woodii. J. Bacteriol. 171:5473-5478. LITERATURE CITED 22. Hsu, T., S. L. Daniel, M. F. Lux, and H. L. 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