Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H 2 -CO 2 -dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H 2 -CO 2 did not replace CO. These results indicated that nitrate blocked the reduction of CO 2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.Most investigations of the CO 2 -fixing acetyl-coenzyme A (CoA) pathway of acetogenic bacteria have focused on the dissimilatory and energy-conserving features of the pathway (13,16,61). However, the anabolic and catabolic functions of the pathway are conceived to be integrated (Fig. 1). The concept that acetogens use the acetyl-CoA pathway for the autotrophic assimilation of carbon is based on a relatively limited number of observations, mainly the absence of other autotrophic CO 2 -assimilating enzymes in Acetobacterium woodii and carbon-labeling patterns obtained with Clostridium thermoaceticum (22, 37). Under heterotrophic conditions (i.e., conditions which do not require obligatory use of the acetyl-CoA pathway for the assimilation of carbon), acetogens may not be strictly dependent on this process, since a number of alternative energy-conserving electron acceptors can be utilized, including fumarate (14, 42), aromatic acrylates (1, 43, 57), inorganic sulfur compounds (2, 3, 27), pyruvate (44), or nitrate (52). Because the use of alternative electron acceptors by acetogens has been examined primarily under heterotrophic conditions, the potential impact of competing reductant sinks on the autotrophic potentials of acetogens is largely unknown. The acetogens C. thermoaceticum (20) and Clostridium thermoautotrophicum (60) are phylogenically very closely related (55) and dissimilate nitrate preferentially to the reduction of CO 2 under heterotrophic conditions (21, 52). The main objective of ...

Nitrate enhanced the vanillin-and vanillate-dependent growth of Clostridium thermoaceticum. Under nitrate-enriched conditions, these aromatic substrates were subject to 0 demethylation. However, acetate, the normal product obtained from 0 demethylation, was not detected. Acetate was also not detected when methanol and CO cultures were supplemented with nitrate; glucose cultures likewise produced approximately one-third less acetate when enriched with nitrate. Reductant derived from the oxidation of these substrates was recovered in nitrite and ammonia. With an ammonia-limited medium employed to evaluate N turnover, the following stoichiometry was observed concomitantly with the consumption of 2.0 mM 0-methyl groups (the recovery of nitrate-derived N approximated 89%): 3.9 mM N03-> 2.8 mM N02-+ 0.7 mM NH3. The results demonstrated that (i) nitrate was preferentially used as an electron sink under conditions that were otherwise acetogenic, (ii) nitrate dissimilation was energy conserving and growth supportive, and (iii) nitrate-coupled utilization of 0-methyl groups conserved more energy than acetogenic 0 demethylation.Carbon dioxide serves as the terminal electron acceptor when acetogenic bacteria use the acetyl coenzyme A (acetylCoA) pathway for a reductant sink (10,11,25,32

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.

Nitrite served as an energy-conserving electron acceptor for the acetogenic bacterium Moorella thermoacetica. Growth occurred in an undefined (0.1% yeast extract) medium containing 20 m M glyoxylate and 5 m M nitrite and was essentially equivalent to that observed in the absence of nitrite. In the presence of nitrite, acetate (the normal product of glyoxylate-derived acetogenesis) was not detected during growth. Instead, growth was coupled to nitrite dissimilation to ammonium, and acetogenesis was limited to the stationary phase. Furthermore, membranes from glyoxylate-grown cells under nitrite-dissimilating conditions were deficient in the b-type cytochrome that is typically found in the membranes of acetogenic cells. Unlike glyoxylate, other acetogenic substrates (fructose, oxalate, glycolate, vanillin, and hydrogen) were not growth supportive in the undefined medium containing nitrite, and glyoxylate-dependent growth did not occur in a nitrite-supplemented, basal (without yeast extract) medium. Glyoxylate-dependent growth by Moorella thermoautotrophica was not observed in the undefined medium containing nitrite.

Glycolate was growth supportive for Moorella thermoacetica ATCC 39073. Growth occurred in undefined and basal culture media containing 20 mM glycolate and was proportional to the concentration of glycolate (10–40 mM). Nitrate inhibited glycolate‐dependent growth in the basal medium. Acetate and cell biomass were the major end products recovered from glycolate. The molar ratio (moles of glycolate required to synthesize a mole of acetate) averaged 1.4 and was in close agreement with the theoretical ratio of 1.3 for glycolate‐derived acetogenesis. Glycolate‐dependent growth yielded approximately 3‐fold less biomass per pair of reducing equivalents utilized than did oxalate‐ or glyoxylate‐dependent growth.