Acetogens utilize the acetyl-CoA Wood-Ljungdahl pathway as a terminal electron-accepting, energy-conserving, CO(2)-fixing process. The decades of research to resolve the enzymology of this pathway (1) preceded studies demonstrating that acetogens not only harbor a novel CO(2)-fixing pathway, but are also ecologically important, and (2) overshadowed the novel microbiological discoveries of acetogens and acetogenesis. The first acetogen to be isolated, Clostridium aceticum, was reported by Klaas Tammo Wieringa in 1936, but was subsequently lost. The second acetogen to be isolated, Clostridium thermoaceticum, was isolated by Francis Ephraim Fontaine and co-workers in 1942. C. thermoaceticum became the most extensively studied acetogen and was used to resolve the enzymology of the acetyl-CoA pathway in the laboratories of Harland Goff Wood and Lars Gerhard Ljungdahl. Although acetogenesis initially intrigued few scientists, this novel process fostered several scientific milestones, including the first (14)C-tracer studies in biology and the discovery that tungsten is a biologically active metal. The acetyl-CoA pathway is now recognized as a fundamental component of the global carbon cycle and essential to the metabolic potentials of many different prokaryotes. The acetyl-CoA pathway and variants thereof appear to be important to primary production in certain habitats and may have been the first autotrophic process on earth and important to the evolution of life. The purpose of this article is to (1) pay tribute to those who discovered acetogens and acetogenesis, and to those who resolved the acetyl-CoA pathway, and (2) highlight the ecology and physiology of acetogens within the framework of their scientific roots.

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N 2 O) and (ii) to characterize the N 2 O-producing bacteria of the earthworm gut. The production of N 2 O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N 2 O were transient products when supplemental nitrate was reduced to N 2 by gut content homogenates. In contrast, nitrite and N 2 O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N 2 O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N 2 O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G؉C contents, and 5 isolates were denitrifiers and reduced nitrate to N 2 O or N 2 . The initial N 2 O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N 2 O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N 2 O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.

The acetogens Sporomusa silvacetica, Moorella thermoacetica, Clostridium magnum, Acetobacterium woodii, and Thermoanaerobacter kivui (i) grew in both semisolid and liquid cultivation media containing O 2 and (ii) consumed small amounts of O 2 . Low concentrations of O 2 caused a lag phase in growth but did not alter the ability of these acetogens to synthesize acetate via the acetyl coenzyme A pathway. Cell extracts of S. silvacetica, M. thermoacetica, and C. magnum contained peroxidase and NADH oxidase activities; catalase and superoxide dismutase activities were not detected.Acetogens have been termed obligate or strict anaerobes. They do not grow aerobically, are isolated mostly from anoxic habitats, and utilize a pathway (the acetyl coenzyme A pathway) that contains enzymes that are extremely sensitive to O 2 (6, 7, 22). However, acetogens (i) can be readily isolated from leaf litter and the mineral soil of well-drained, oxic soils (8, 9), (ii) can tolerate periods of oxygenation in soils (21), (iii) are active in termite guts that have steep oxygen gradients (20), and (iv) occur in high numbers in transiently oxygenated rhizosphere sediments colonized by sea grass (12). These observations suggest that certain acetogens must cope with O 2 under in situ conditions. In preliminary studies, the classic acetogen Moorella thermoacetica was found to reduce resazurin in O 2 -supplemented medium (A. Gößner and H. L. Drake, unpublished data), and the objective of the present study was to determine the tolerance and metabolic response of model acetogens toward O 2 (for a preliminary report of this study, see A. Karnholz, K. Küsel, and H. L. Drake, Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, abstr. I-91, p. 401, 2000.Organisms, media, and growth conditions. The acetogens used in this study were selected because each is a well-described model acetogen that has been isolated from a different habitat. The temperatures of incubation for Sporomusa silvacetica (DSM 10669; isolated from soil), M. thermoacetica (DSM 1974; isolated from horse manure), Clostridium magnum (DSM 2767; isolated from fresh water sediment), Acetobacterium woodii (DSM 1030; isolated from a marine estuary), and Thermoanaerobacter kivui (DSM 2030; isolated from lake sediment) were 30, 55, 30, 30, and 55°C, respectively. The acetogens were cultivated in a carbonate-buffered, undefined (U) medium containing yeast extract, vitamins, and trace metals but no reducing agents (4). Medium was dispensed under CO 2 into 27-ml crimp seal culture tubes (7 ml of medium per tube) or 1-liter infusion bottles (500 ml of medium per bottle; used for preparation of cell extracts), which were then sealed and autoclaved; the pH was approximately 6.7. Anoxic aqueous stock solutions of glucose or fructose (prepared under argon) were filter sterilized and added to the medium by syringe injection by using O 2 -free techniques. Culture tubes and bottles containing liquid medium were incubated in a horizontal, static position. Culture tubes were shaken vigorously before opt...

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

Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum, and Sporomusa silvacetica were found to contain carbonic anhydrase (CA). Minimal to no CA activity was detected in Moorella thermoautotrophica, Moorella thermoacetica subsp. "pratumsolum," Sporomusa termitida, and Thermoanaerobacter kivui. Of the acetogens tested, A. woodii had the highest CA specific activity, approximately 14 U mg of protein ؊1 , in extracts of either glucose-or H 2 -CO 2 -cultivated cells. CA of A. woodii was cytoplasmic and was purified approximately 300-fold to a specific activity of 5,236 U mg of protein ؊1