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 acetate concentrations per unit of biomass synthesized than did CO-or glucose-dependent growth. In dated the minimal nutritional requirements of this acetogen (37) so that a definitive assessment could be made of its heterotrophic and chemolithotrophic potentials. In the study presented here, numerous strains of C. thermoaceticum were obtained from various sources and evaluated. In addition, Acetogenium kivui (33, 34), a thermophilic nonclostridial acetogen which is capable of H2-dependent chemolithotrophic growth, was also included in this evaluation and used for comparative purposes. In this report, we demonstrate for the first time that certain strains of C. thermoaceticum grow chemolithotrophically (requiring only trace levels of nicotinic acid as the sole vitamin) at the expense of H2 or CO. Besides the overall metabolic properties exhibited by C. thermoaceticum and A. kivui under chemolithotrophic conditions, evidence is also presented which suggests that the type of energy source used during growth (e.g., H2 versus glucose) influences the expression or activity of hydrogenase and CO dehydrogenase in both of these acetogens. MATERIALS AND METHODSBacterial strains and cultivation. C. thermoaceticum (see Table 1 for strains used in this study) and A.

Clostridium thermoaceticum ATCC 39073 converted vanillate to catechol. Although carboxylated aromatic compounds which did not contain methoxyl groups were not by themselves growth supportive, protocatechuate and p-hydroxybenzoate (nonmethoxylated aromatic compounds) were converted to catechol and phenol, respectively, during carbon monoxide-dependent growth. Syringate is not subject to decarboxylation by C. thermoaceticum (Z. Wu, S. L. Daniel, and H. L. Drake, J. Bacteriol. 170:5705-5708, 1988), and sustained growth at the expense of syringate-derived methoxyl groups was dependent on supplemental CO2. In contrast, vanillate was growth supportive in the absence of supplemental CO2, and 14CO2 was the major 14C-labeled product during [carboxyl-14C]vanillate-dependent growth. Furthermore, the decarboxylation of protocatechuate and p-hydroxybenzoate supported methanol- and 1,2,3-trimethoxybenzene-dependent growth (CO2 is required for growth at the expense of these substrates) when supplemental CO2 was depleted from the growth medium, and the decarboxylation of protocatechuate was concomitant with improved cell yields of methanol cultures. These findings demonstrate that (i) C. thermoaceticum is competent in the decarboxylation of certain aromatic compounds and (ii) under certain conditions, decarboxylation may be integrated to the flow of carbon and energy during acetogenesis.

: 1990). By using 4-hydroxybenzoate as a model substrate, an assay was devel9ped to study the expression apd activity of the decarboxylase involved in the activa'tion of aromatic carboxyl groups. The aromatic-dppendent decarboxylase was induced by carboxylated aromatic compounds in the early'stages of'growth and was not repressed by glucose or. other acetogenic substrates; nonutilizable carboxylated aromatic compounds did not induce the decarboxylase. The decarboxylase activity displayed saturation kinetics at both whole-cell and cell extract levels, was sensitive to oxidation, and was not affected by exogenous energy sources. However, at the whole-cell level, metabolic inhibitors decreased the decarboxylase activity. Supplemental biotin or avidin did not significantly affect decarboxylation. The aromatic-dependent decarboxylase was specific for benzoates with a hydroxyl group in the para position of the aromatic ring; the meta position could bie occupied by various substituent groups (-H, -OH, -OCH3, -Cl, or -F). The carboxyl carbon from [carboxyl-'4C]vaniliate went primarily to 14C02 in short-term decarboxylase assays. During growth, the aromatic carboxyl group went primarily to CO2 under C02-enriched conditions. However, under C02-limited conditions, the aromatic carboxyl carbon went nearly totally to acetate, with equal distribution between the carboxyl and methyl carbons, thus demonstrating that acetate could be totally synthesized from aromatic carboxyl groups. In contrast, when cocultivated (i.e., supplemented) with CO under C02-limited conditions, the aromatic carboxyl group went primarily to the methyl carbon of acetate.

Vanillin was subjet to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO‐dependent growth of Peptostreptococcus productus. the aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co‐metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO‐dependent growth, C. thermoaceticum reduced 4‐hydroxybenzaldehyde to 4‐hydroxylbenzyl alcohol while P. productus both reduced and oxidized 4‐hydroxybenzaldehyde to 4‐hydroxybenzyl alcohol and 4‐hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiC12. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the midto late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 ,uM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose-and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.