Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.

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Chain elongation into medium-chain carboxylates, such as n-caproate and n-caprylate, with ethanol as an electron donor and with open cultures of microbial consortia (i.e., reactor microbiomes) under anaerobic conditions is being developed as a biotechnological production platform. The goal is to use the high thermodynamic efficiency of anaerobic fermentation to convert organic biomass or organic wastes into valuable biochemicals that can be extracted. Several liter-scale studies have been completed and a first pilot-plant study is underway. However, the underlying microbial pathways are not always well understood. In addition, an interdisciplinary approach with knowledge from fields ranging from microbiology and chemical separations to biochemistry and environmental engineering is required. To bring together research from different fields, we reviewed the literature starting with the microbiology and ending with the bioprocess engineering studies that already have been performed. Because understanding the microbial pathways is so important to predict and steer performance, we delved into a stoichiometric and thermodynamic model that sheds light on the effect of substrate ratios and environmental conditions on product formation. Finally, we ended with an outlook.

The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.

Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions. butyryl-CoA dehydrogenase ͉ electron transfer flavoproteins ͉ genome sequence ͉ Rnf-dependent energy conservation

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)-and ferredoxin-dependent formation of H 2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E 0 ‫؍‬ ؊410 mV) with NADH (E 0 ‫؍‬ ؊320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E 0 ‫؍‬ ؊10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH ؉ 1 oxidized ferredoxin ؉ 1 crotonyl-CoA ‫؍‬ 2 NAD ؉ ؉ 1 ferredoxin reduced by two electrons ؉ 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.Clostridium kluyveri is a strictly anaerobic gram-positive endospore-forming bacterium (2). Among the clostridia this organism is unique in fermenting ethanol and acetate to butyrate, caproate, and H 2 (38, 49) and in deriving a large portion (30%) of its cell carbon from CO 2 (52). Both its energy metabolism and its pathways of biosynthesis have therefore been the subject of many investigations (26, 39a). In particular, understanding the energy metabolism of C. kluyveri remains a challenge for microbiologists, and one of the pertinent questions is how this organism generates H 2 (36, 39a, 48).

Energy conservation in chemotrophic organisms is generally coupled to redox reactions in catabolic pathways. In the oxidative part or branch, "energy-rich" compounds are formed, from which ATP is generated via substrate-level phosphorylation (SLP). In the reductive branch the electron carriers are reoxidized by a terminal acceptor; in this way an electrochemical ion gradient (⌬H ϩ or ⌬Na ϩ ) at the cytoplasmic membrane is established, which is used for ATP synthesis, transport across membranes, and motility. This second type of energy conservation is called respiration or electron transport phosphorylation (ETP). Bacterial fermentations are considered apparent exceptions to this generalization because they are thought to lack ETP (41). In these fermentations the substrate serves not only as an electron donor but also as a terminal acceptor, since oxygen, nitrate, fumarate, etc. are absent (44). An example is the fermentation of glutamate via 3-methylasparate by the closely related anaerobic bacteria Clostridium tetani, Clostridium tetanomorphum, and Clostridium pascui to ammonia, acetate, butyrate, and molecular hydrogen according to equation 1 (Fig. 1) (8,16,51):In the first part of this pathway, glutamate is converted to ammonia, acetate, and pyruvate, which is oxidized by ferredoxin and coenzyme A (CoA) to acetyl-CoA and CO 2 . In order to regenerate the oxidant, acetyl-CoA and protons are reduced to butyryl-CoA and hydrogen, respectively. From 2 butyrylCoA and 1 acetyl-CoA, 3 ATP are obtained via SLP. But the free enthalpy required to synthesize 1 ATP (Ϫ317/3 ϭ Ϫ106 kJ mol Ϫ1 ) is still much higher than that needed in other systems; i.e., the efficiency is low ( ϭ 42%). Usually, Ϫ75 Ϯ 5 kJ mol Ϫ1 ( ϭ 60%) is considered the minimum free enthalpy for ATP synthesis (44). It should be noted that without hydrogen formation the ATP yield would drop to 2.5 mol ATP (5 mol glutamate)Ϫ1 , because in order to maintain the redox balance, acetyl-CoA has to be completely reduced to butyrate. Thus, hydrogen formation increases SLP. If all the reducing equivalents dissipated as hydrogen and no butyrate was formed, the yield would rise to 5 mol ATP (5 mol glutamate)Ϫ1 . This, however, is thermodynamically not possible (Ϫ46 kJ mol glutamate Ϫ1 ). The oxidation of pyruvate derived from glutamate yields reduced ferredoxin (E 0 Ј Յ Ϫ420 mV), whereas NADH (E 0 Ј ϭ Ϫ320 mV) is the reductant in butyrate synthesis. The difference (Ն100 mV) could be used for additional energy conservation. Recently, we discovered the enzyme that catalyzes the reduction of NAD ϩ with reduced ferredoxin (Fig. 1) and characterized it as an Rnf-type NADH ferredoxin oxidoreductase localized in the membrane of C. tetanomorphum. Although it has not been demonstrated yet that this clostridial Rnf protein pumps protons or sodium ions, its cellular localization and the homology of four of its six subunits with four of the subunits of NADH-quinone reductase (Nqr) from Vibrio alginolyticus and Vibrio cholerae strongly suggest that this enzyme is involved in ener...

The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction.